U.S. patent number 9,233,370 [Application Number 13/204,121] was granted by the patent office on 2016-01-12 for magnetic immunosensor and method of use.
This patent grant is currently assigned to ABBOTT POINT OF CARE INC.. The grantee listed for this patent is Jinghua Hu, Cary James Miller. Invention is credited to Jinghua Hu, Cary James Miller.
United States Patent |
9,233,370 |
Miller , et al. |
January 12, 2016 |
Magnetic immunosensor and method of use
Abstract
The present invention provides apparatus and methods for the
rapid determination of analytes in liquid samples by immunoassays
incorporating magnetic capture of beads on a sensor capable of
being used in the point-of-care diagnostic field.
Inventors: |
Miller; Cary James (Ottawa,
CA), Hu; Jinghua (Ottawa, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Miller; Cary James
Hu; Jinghua |
Ottawa
Ottawa |
N/A
N/A |
CA
CA |
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Assignee: |
ABBOTT POINT OF CARE INC.
(Princeton, NJ)
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Family
ID: |
44511560 |
Appl.
No.: |
13/204,121 |
Filed: |
August 5, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20120034633 A1 |
Feb 9, 2012 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61371109 |
Aug 5, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
27/745 (20130101); G01N 33/54326 (20130101); B01L
3/502761 (20130101); G01N 33/5438 (20130101); B01L
2200/0684 (20130101); B01L 2300/0645 (20130101); B01L
2400/043 (20130101); B01L 2400/0688 (20130101); B01L
2300/0816 (20130101); B01L 2400/0481 (20130101); B01L
2400/0683 (20130101) |
Current International
Class: |
G01N
33/543 (20060101); B01L 3/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 00/51814 |
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Sep 2000 |
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WO |
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WO 01/87458 |
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Nov 2001 |
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WO |
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Other References
Laurrell et al., Methods in Enzymology, vol. 73,
"Electroimmunoassay", Academic Press, New York, 339, 340, 346-348
(1981). cited by applicant .
M.J. Green (1987) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 316:
135-142. cited by applicant .
Bruls et al., Lab Chip 9, 3504-3510 (2009) (Abstract only). cited
by applicant .
Dittmer et al., Clin. Chim. Acta (2010), doi:
10.1016/j.cca.2010.03.001 (Abstract only). cited by applicant .
Peng et al., "A bead-based electrochemical biosensor with
integrated magnetic manipulation for controllable sample
preconcentration", 15.sup.th International Conf. on Solid-State
Sensors, Actuators and Microsystems, Transducers 2009 IEEE, pp.
1802-1805. cited by applicant .
International Search Report and Written Opinion for
PCT/US2011/046757 mailed Oct. 12, 2011. cited by applicant .
Estes et al., "On chip cell separator using magnetic bead-based
enrichment depletion of various surface markers", Biomed
Microdevices (2009), 11: 509-515. cited by applicant .
Berti et al., "Microfluidic-based electrochemical genosensor
coupled to magnetic beads for hybridization detection", vol. 77,
No. 3, Jan. 15, 2009, pp. 971-978. cited by applicant .
International Search Report and Written Opinion for
PCT/US2011/046753 mailed Dec. 30, 2011. cited by applicant .
International Search Report and Written Opinion for
PCT/US2011/046758 mailed Oct. 7, 2011. cited by applicant .
International Search Report and Written Opinion for
PCT/US2011/046761 mailed Oct. 7, 2011. cited by applicant .
International Preliminary Report on Patentability mailed Feb. 14,
2013 in corresponding International Application No.
PCT/US2011/046757. cited by applicant .
Rossier, et al., "Plasma Etched Polymer Microelectrochemical
Systems," Lab Chip, 2002, 2, pp. 145-150. cited by applicant .
Rossier, et al., Journal of Laboratory Automation, Dec. 2008, vol.
13, No. 6, pp. 322-329. cited by applicant .
Ramanujan, "Magnetic Particles for Biomedical Applicatoins,"
Chapter 17, Biomedical Materials, Springer, NY, 2009. cited by
applicant .
Kuhn, et al., "Developing Multiplexed Assays for Troponin I and
Interleukin-33 in Plasma by Peptide Immunoaffinity Enrichment and
Targeted Mass Spectrometry," Clinical Chemistry, vol. 55, No. 6,
pp. 1108-1117. cited by applicant .
Office Action for corresponding U.S. Appl. No. 13/204,172 dated
Jun. 5, 2014. cited by applicant .
Final Office Action for corresponding U.S. Appl. No. 13/204,109
dated Jun. 18, 2014. cited by applicant .
K&J Magnetics (kjmagnetics.com, neodymium cylinder magnets,
D22-N52). cited by applicant .
Office Action for corresponding European Appl. No. 11748815.5 dated
Aug. 20, 2014. cited by applicant .
Office Action for Chinese Appl. No. 201180048048.4 dated Sep. 3,
2014. cited by applicant.
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Primary Examiner: Brown; Melanie Y
Assistant Examiner: Martinez; Rebecca
Attorney, Agent or Firm: Kilpatrick Townsend Stockton
LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
The present invention claims priority to U.S. Provisional
Application No. 61/371,109, filed on Aug. 5, 2010, the entire
contents and disclosure of which are hereby incorporated by
reference.
Claims
What is claimed is:
1. A microfabricated magnetic layer on a substantially planar
surface, wherein the microfabricated magnetic layer comprises:
high-field permanent magnetic particulates dispersed in a
thermally, chemically or photoformably cured immobilization matrix;
wherein the immobilization matrix comprises a polyimide, polyvinyl
alcohol, or epoxy resin; wherein the microfabricated magnetic layer
is positioned substantially proximate to a microfabricated sensing
electrode, wherein the electrode is connected by a conductive line
to instrument electronics, wherein the microfabricated magnetic
layer is not the conductive line, wherein the microfabricated
magnetic layer is positioned to attract magnetically susceptible
beads coated with a capture antibody to a target analyte, wherein
the electrode is an amperometric electrode, and wherein the
magnetic layer is configured to concentrate the magnetically
susceptible beads with the capture antibody and the target analyte
within a sample fluid at the electrode for measurement, and wherein
the amperometric electrode is configured to detect the target
analyte.
2. The microfabricated magnetic layer of claim 1, wherein the
magnetic particulates comprise a neodymium iron boron (NdFeB)
alloy.
3. The microfabricated magnetic layer of claim 1, wherein the
magnetic particulates comprise a Nd.sub.2Fe.sub.14B alloy.
4. The microfabricated magnetic layer of claim 1, wherein the
magnetic particulates have an average particle size of from 0.01
.mu.m to 20 .mu.m.
5. The microfabricated magnetic layer of claim 1, wherein the
magnetic particulates have an average particle size of from 0.01
.mu.m to 5 .mu.m.
6. The microfabricated magnetic layer of claim 1, wherein the
immobilization matrix comprises polyimide.
7. The microfabricated magnetic layer of claim 1, wherein the
immobilization matrix comprises polyvinyl alcohol.
8. The microfabricated magnetic layer of claim 1, wherein the
magnetic particulates provide a magnetic field of greater than
about 0.1 Tesla.
9. The microfabricated magnetic layer of claim 1, wherein the
microfabricated magnetic layer is positioned to yield an event
horizon for the magnetically susceptible beads in the range of less
than about 200 .mu.m in the region of the electrode.
10. The microfabricated magnetic layer of claim 1, wherein the
electrode is a gold microarray.
11. The microfabricated magnetic layer of claim 1, wherein the
microfabricated magnetic layer is positioned over the substantially
planar surface.
12. The microfabricated magnetic layer of claim 1, wherein the
microfabricated magnetic layer is directly attached to the
substantially planar surface.
13. The microfabricated magnetic layer of claim 1, wherein the
microfabricated magnetic layer is coated onto the surface of the
substantially planar surface.
14. The microfabricated magnetic layer of claim 1, wherein the
microfabricated magnetic layer is patterned onto the surface of the
substantially planar surface.
15. The microfabricated magnetic layer of claim 1, wherein the
microfabricated magnetic layer is positioned proximate to the
sensing electrode.
Description
FIELD OF THE INVENTION
The present invention generally relates to an apparatus and method
for rapid determination of analytes in liquid samples by
immunoassays incorporating magnetic capture of beads on a sensor,
capable of being used in the point-of-care diagnostic field,
including, for example, use at accident sites, emergency rooms, in
surgery, in intensive care units, and also in non-medical
environments.
BACKGROUND OF THE INVENTION
A multitude of laboratory immunoassay tests for analytes of
interest are performed on biological samples for diagnosis,
screening, disease staging, forensic analysis, pregnancy testing
and drug testing, among others. While a few qualitative tests, such
as pregnancy tests, have been reduced to simple kits for a
patient's home use, the majority of quantitative tests still
require the expertise of trained technicians in a laboratory
setting using sophisticated instruments. Laboratory testing
increases the cost of analysis and delays the patient's receipt of
the results. In many circumstances, this delay can be detrimental
to the patient's condition or prognosis, such as for example the
analysis of markers indicating myocardial infarction and heart
failure. In these and similar critical situations, it is
advantageous to perform such analyses at the point-of-care,
accurately, inexpensively and with minimal delay.
Many types of immunoassay devices and processes have been
described. For example, a disposable sensing device for measuring
analytes by means of immunoassay in blood is disclosed by Davis et
al. in U.S. Pat. No. 7,419,821. This device employs a reading
apparatus and a cartridge that fits into the reading apparatus for
the purpose of measuring analyte concentrations. A potential
problem with such disposable devices is variability of fluid test
parameters from cartridge to cartridge due to manufacturing
tolerances or machine wear. U.S. Pat. No. 5,821,399 to Zelin
discloses methods to overcome this problem using automatic flow
compensation controlled by a reading apparatus having
conductimetric sensors located within a cartridge. Each of these
patents is hereby incorporated by reference in their respective
entireties.
Electrochemical detection, in which the binding of an analyte
directly or indirectly causes a change in the activity of an
electroactive species adjacent to an electrode, has also been
applied to immunoassays. For an early review of electrochemical
immunoassays, see Laurell et al., Methods in Enzymology, vol. 73,
"Electroimmunoassay", Academic Press, New York, 339, 340, 346-348
(1981).
In an electrochemical immunosensor, the binding of an analyte to
its cognate antibody produces a change in the activity of an
electroactive species at an electrode that is poised at a suitable
electrochemical potential to cause oxidation or reduction of the
electroactive species. There are many arrangements for meeting
these conditions. For example, electroactive species may be
attached directly to an analyte, or the antibody may be covalently
attached to an enzyme that either produces an electroactive species
from an electroinactive substrate or destroys an electroactive
substrate. See, M. J. Green (1987) Philos. Trans. R. Soc. Lond. B.
Biol. Sci. 316:135-142, for a review of electrochemical
immunosensors. Magnetic components have been integrated with
electrochemical immunoassays. See, for example, U.S. Pat. Nos.
4,945,045; 4,978,610; and 5,149,630, each to Forrest et al.
Furthermore, jointly-owned U.S. Pat. No. 7,419,821 to Davis et al.
(referenced above) and U.S. Pat. Nos. 7,682,833 and 7,723,099 to
Miller et al. teach immunosensing with magnetic particles.
Microfabrication techniques (e.g., photolithography and plasma
deposition) are attractive for construction of multilayered sensor
structures in confined spaces. Methods for microfabrication of
electrochemical immunosensors, for example on silicon substrates,
are disclosed in U.S. Pat. No. 5,200,051 to Cozette et al., which
is hereby incorporated in its entirety by reference. These include
dispensing methods, methods for attaching biological reagent, e.g.,
antibodies, to surfaces including photoformed layers and
microparticle latexes, and methods for performing electrochemical
assays.
U.S. Pat. No. 7,223,438 to Mirkin et al. describes a method of
forming magnetic nanostructures by depositing a precursor onto a
substrate using a nanoscopic tip, and then converting the precursor
to form a magnetic nanostructure. U.S. Pat. No. 7,106,051 to Prins
et al. describes a magnetoresistive sensing device for determining
the density of magnetic particles in a fluid.
U.S. Pat. Appl. Pub. 2009/0191401 to Deetz et al. is directed to
magnetic receptive paints and coatings that allow magnets to stick
to coated surfaces. These paint and coating compositions contain
multiple-sized ferromagnetic particles and a base resin with
minimal or no fillers and provide an ultra smooth finish on a
substrate. U.S. Pat. No. 5,587,102 to Stern et al. discloses a
latex paint composition comprising iron particles and U.S. Pat. No.
5,843,329 to Deetz provides techniques for blending magnetic
receptive particles into solution for making magnetic coatings.
Jointly-owned U.S. Pat. Nos. 5,998,224 and 6,294,342 to Rohr et al.
disclose assay methods utilizing the response of a magnetically
responsive reagent to influence a magnetic field to qualitatively
or quantitatively measure binding between specific binding pair
members. Each of these patents is hereby incorporated by reference
in its entirety.
Both an integrated biosensor for multiplexed immunoassays based on
actuated magnetic nanoparticles and a high sensitivity
point-of-care test for cardiac troponin based on an optomagnetic
biosensor have been described. See, Bruls et al., Lab Chip 9,
3504-3510 (2009) and Dittmer et al., Clin. Chim. Acta (2010),
doi:10.1016/j.cca.2010.03.001,respectively. There are numerous
disclosures of the use of magnetically susceptible particles, e.g.,
U.S. Pat. No. 4,230,685 to Senyei et al., U.S. Pat. No. 4,554,088
to Whitehead et al., and U.S. Pat. No. 4,628,037 to Chagnon et al.
An important factor in the use of these particles in assays is
efficient mixing to enhance the reaction rate between the target
analyte and the particle surfaces, as opposed to the use of a
macro-binding surface that mainly relies on diffusion. Magnetic
mixing systems are disclosed in U.S. Pat. No. 6,231,760 to Siddiqi
and U.S. Pat. No. 6,764,859 to Kreuwel et al.
Notwithstanding the above literature, there remains a need in the
art for improved immunosensing devices with greater sensitivity for
the detection of analytes, including, for example, cardiac troponin
I for early detection of myocardial infarction. These and other
needs are met by the present invention as will become clear to one
of skill in the art to which the invention pertains upon reading
the following disclosure.
SUMMARY OF THE INVENTION
The present invention is directed to a magnetic immunosensing
device and methods of performing an immunoassay with magnetic
immunosensors to provide diverse real-time or near real-time
analysis of analytes.
In one embodiment, the invention is directed to a magnetic
immunosensing device, comprising: a sensing electrode on a
substantially planar chip, wherein the electrode is positioned in a
conduit for receiving a sample mixed with antibody-labeled
magnetically susceptible beads; and an integrated high-field
permanent magnetic layer on the chip, wherein the magnetic layer is
positioned relative to the electrode, thereby attracting the beads
substantially proximate to the electrode and substantially
retaining the beads at the electrode surface during removal of
unbound sample and washing of the electrode.
Another embodiment of the present invention is directed to a
microfabricated magnetic layer on a substantially planar surface,
comprising: high-field permanent magnetic particulates, wherein
said particulates are dispersed in a thermally, chemically or
photoformably cured immobilization matrix; and a microfabricated
sensing electrode.
In another embodiment, a method of performing a sandwich
immunoassay for an analyte in a sample with a magnetic
immunosensor, wherein said immunosensor comprises a sensing
electrode on a substantially planar chip and an integrated layer on
said chip that is magnetized and positioned substantially proximate
to the electrode is provided. This method comprises (a) mixing
magnetically susceptible beads coated with a capture antibody to an
analyte with a sample containing the analyte and a signal antibody
to form a sandwich on said beads; (b) applying the mixture to the
immunosensor; (c) magnetically localizing and retaining at least a
portion of said beads on the electrode; (d) washing the unbound
sample from the electrode; (e) exposing the signal antibody of the
sandwich to a signal generating reagent; and (f) measuring a signal
from the reagent at the electrode.
In a further embodiment, the invention is directed to a method of
performing a competitive immunoassay for an analyte in a sample
with a magnetic immunosensor, wherein said immunosensor comprises a
sensing electrode on a substantially planar chip and an integrated
layer on said chip that is magnetized and positioned substantially
proximate to said electrode. This method comprises (a) mixing
magnetically susceptible beads coated with a capture antibody with
a sample containing a first analyte and a second analyte, wherein
the second analyte is labeled, to permit binding on said beads; (b)
applying the mixture to the immunosensor; (c) magnetically
localizing and retaining at least a portion of the beads on the
electrode; (d) washing the unbound sample from the electrode; (e)
exposing the second analyte to a signal generating reagent; and (f)
measuring a signal from said reagent at the electrode.
The above summary of the present invention is not intended to
describe each illustrated embodiment or every implementation of the
present invention. The figures and the detailed description that
follow more particularly exemplify these embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other objectives, features and advantages of the present
invention are described in the following detailed description of
the specific embodiments and are illustrated in the following
Figures, in which:
FIG. 1 is a cross-sectional illustration of a grooved immunosensor
chip in a conduit with applied magnetic fields in accordance with
one embodiment of the present invention;
FIG. 2 is a top view of the grooved immunosensor chip and a
microelectrode array;
FIGS. 3A-C illustrate various exemplary configurations for the
positioning of a rare earth permanent magnet below an immunosensor
chip within a cartridge;
FIG. 4 is a schematic of magnetic field lines for sensor
configurations;
FIG. 5 is a micrograph of magnetically susceptible beads captured
on a chip surface where the center of the magnet is positioned
directly below the perimeter of an immunosensor;
FIG. 6 illustrates three traces of the electrochemical detection
step for the magnetically susceptible bead capture assay: plasma
with zero cTnI; Cliniqa control level 3; and Cliniqa control level
4.
FIG. 7 illustrates the positioning of a rare earth permanent magnet
below an immunosensor chip within a cartridge housing in accordance
with one embodiment of the present invention;
FIG. 8 illustrates a foldable cartridge housing in accordance with
one embodiment of the present invention where the rare earth
permanent magnet is positioned underneath an immunosensor chip;
FIG. 9 is a micrograph of magnetically susceptible beads localized
on a patterned magnetic layer of PVA and NdFeB particles;
FIG. 10 is a micrograph of a fractured cross-section of the device
of FIG. 9;
FIGS. 11A-C include micrographs of a patterned PVA film with
various particle sizes of NdFeB (FIG. 11A); ground 6 .mu.m MQP in
polyimide (FIG. 11B); ground 6 .mu.m MQP in polyimide (FIG.
11C);
FIG. 12A is a micrograph of a 6 .mu.m MQP NdFeB powder and FIG. 12B
is a micrograph of the 6 .mu.m MQP NdFeB powder comminuted using a
ball mill;
FIG. 13 is a micrograph of beads captured on NdFeB particle
surfaces;
FIGS. 14A and 14B show exemplary base immunosensor electrode arrays
partially covered with a printed NdFeB magnetic layer leaving a
portion of the perimeter of the array exposed;
FIGS. 15A and 15B depict the over-printed magnetic layer in
accordance with other embodiments of the present invention;
FIG. 16 is a sheared sensor illustrating the printed magnetic layer
profile of FIGS. 15A and 15B;
FIG. 17 illustrates the etched trench process in accordance with
one embodiment of the present invention;
FIG. 18 is a top view of an exemplary underside trench design
etched into a silicon wafer;
FIG. 19 is the cross-sectional profile of the underside trench;
FIGS. 20A and 20B depict different views of the etched trench;
FIG. 21 shows the etched trench filled with NbFeB powder in a
polyimide resin;
FIGS. 22A and 22B are micrographs of a rectangular trench produced
on a silicon substrate via reactive ion etching: FIG. 22A shows a
cross-section of the trench and FIG. 22B shows a different
cross-section of the trench filled with NbFeB powder in a polyimide
resin;
FIG. 23 depicts an exemplary combined sensor design for an expanded
detection range where the magnetic zone is comprised a screen
printed line of NdFeB powder in a polyimide matrix;
FIG. 24 depicts an exemplary combined sensor design for an expanded
detection range where the magnetic zone is comprised of a bulk
NdFeB magnet;
FIG. 25 depicts another combined sensor design in accordance with
one embodiment of the present invention where the open circles on
the chip are potential print locations for the reagents of the
present invention;
FIG. 26 is a schematic of an oscillating bead immunoassay (OBIA)
with a central immunosensor flanked by two adjacent magnetic zones
with the small bead moving in-between in accordance with one
embodiment of the present invention;
FIG. 27 is a schematic illustrating the degree of bead capture with
time;
FIG. 28 shows a multiplexed OBIA where several different types of
analyte-capturing beads are present, but where they are effectively
separated onto their individual capture sites;
FIG. 29 illustrates the comparative principle of an amperometric
immunoassay for determination of troponin I (TnI), a marker of
cardiac injury;
FIG. 30 is an isometric top view of an immunosensor cartridge cover
of one embodiment of the invention;
FIG. 31 is an isometric bottom view of an immunosensor cartridge
cover of one embodiment of the invention;
FIG. 32 is a top view of the layout of a tape gasket for an
immunosensor cartridge of one embodiment of the invention;
FIG. 33 is an isometric top view of an immunosensor cartridge base
of one embodiment of the invention;
FIG. 34 illustrates an exemplary segment forming means;
FIG. 35 is a top view of one embodiment of an immunosensor
cartridge; and
FIG. 36 is a schematic view of the fluidics of one embodiment of an
immunosensor cartridge.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an apparatus and its method of use
for determining the presence or concentrations of analytes in a
liquid sample with a single-use disposable cartridge. The invention
is adapted for conducting diverse real-time or near real-time
assays of analytes. This invention is particularly relevant to high
sensitivity cardiac troponin assays in whole blood samples.
In specific embodiments, the invention relates to the determination
of analytes in biological samples such as blood using magnetic
electrochemical immunosensors or other ligand/ligand receptor-based
biosensors based on a magnetically susceptible bead localization
step. The present invention also hereby incorporates by reference
in their respective entireties jointly-owned U.S. Pat. No.
7,419,821 to Davis et al. and U.S. Pat. Nos. 7,682,833 and
7,723,099 to Miller et al., each of which is referenced above.
One notable advantage of the magnetically susceptible bead capture
approach of the present invention is in improving the low-end
sensitivity of immunoassays where there is a perceived benefit in
detection of extremely low levels of a marker of myocardial injury
(e.g., cTnI). Further advantages and benefits of the embodiments of
the invention described and disclosed herein include but are not
limited to ease of use, automation of many, if not all steps, of
the analysis and elimination of user induced error in the
analysis.
I. Magnetic Immunosensor
Various embodiments of the present invention are directed to a
magnetic immunosensing device or immunosensor that includes a base
sensor or sensing electrode on a substantially planar chip where
the sensing electrode is positioned in a conduit for receiving a
sample mixed with beads that can be attracted to a magnet, or
respond to a magnetic field.
A high-field magnet, e.g., a permanent magnet or an electromagnet,
is positioned proximate to the immunosensor chip (e.g., below) or
incorporated into the immunosensor chip, for attracting the beads
in the conduit substantially proximate to the sensing electrode.
This magnetic zone functions to substantially retain the beads at
or near the sensing electrode surface during removal of the unbound
sample and washing of the electrode. As described in detail herein,
the beads are coated with an antibody to an analyte in said sample,
which provides the basis for an immunoassay. In preferred
embodiments, a system comprising a reading apparatus or reader and
a single-use cartridge containing the magnetic immunosensor and all
the other assay components is used to analyze an analyte in a
biological sample.
A. High-Field Magnet
In certain embodiments of the invention, the magnetic immunosensor
comprises a sensing electrode on a substantially planar chip and
has a high-field magnet, e.g., a permanent magnet or an
electromagnet, positioned proximate to (e.g., below) or associated
with the chip. The magnetic immunosensor of the present invention
provides a field of greater than about 0.1 Tesla and has an "event
horizon" (as defined herein) that can efficiently draw beads from a
range of about 0.05 mm to about 5 mm in the region of the sensing
electrode.
The high-field magnet, e.g., permanent magnet or electromagnet, of
the present invention includes any material that provides a high
magnetic field (e.g., greater than about 0.1 Tesla, greater than
0.4 Tesla or greater than 1 Tesla). The magnetic field can be
measured, for example, as the remnant field on a substantially flat
surface area of a magnet. While the preferred material is a
neodymium iron boron alloy (NdFeB) alloy, and more preferably
Nd.sub.2Fe.sub.14B, other materials may be used. For example, those
skilled in the art will recognize that high-field permanent magnets
can include ferrite or aluminum nickel cobalt (AlNiCo) magnets,
which typically exhibit fields of 0.1 to 1 Tesla. Other high-field
permanent magnets comprised of alloys of rare earth elements (e.g.,
neodymium alloys and samarium cobalt (SmCo) alloys) exhibit fields
in excess of 1 Tesla, e.g., greater than 1.2 Tesla or greater than
1.4 Tesla.
Rare earth magnets are generally brittle and also vulnerable to
corrosion, and as such these materials are frequently plated or
coated to protect them from breaking and chipping. In addition, the
Curie point of rare earth magnets is substantially above the
temperatures encountered in the immunoassay of the present
invention, which may be run in the ambient to about 50.degree. C.
range, typically thermostated at 37.degree. C. for assays in
blood.
As used herein, "Curie point" or "Curie temperature" refers to a
characteristic property of a ferromagnetic material. The Curie
point of a ferromagnetic material is the temperature above which it
loses its characteristic ferromagnetic ability to possess a net
(spontaneous) magnetization in the absence of an external magnetic
field. At temperatures below the Curie point, the magnetic moments
are partially aligned within magnetic domains in ferromagnetic
materials. As the temperature is increased from below the Curie
point, thermal fluctuations increasingly destroy this alignment,
until the net magnetization becomes zero at and above the Curie
point. Above the Curie point, the material is purely
paramagnetic.
In another embodiment, the high-field magnet comprises an
electromagnet in which the magnetic field is produced by the flow
of electric current. The electric current may be provided by a
reader, in which the immunosensing device is inserted and with
which the immunosensing device is in electrical contact.
1. Bulk Magnet Positioned in Housing of Magnetic Immunosensor
The magnetic immunosensor of some embodiments of the invention
comprises a sensing electrode on a substantially planar chip and a
bulk permanent high-field magnet positioned proximate to the
electrode (e.g., below or on the opposite side of the chip). In
certain preferred embodiments, the bulk permanent high-field magnet
is positioned in the housing (e.g., cut out or trench in the
plastic cartridge) of the magnetic immunosensing device.
Preferably, the bulk permanent high-field magnet is positioned
within the base of the plastic cartridge housing (e.g.,
non-coplanar with the sensing electrode). In other embodiments, the
magnet is positioned adjacent to or within the reading apparatus or
reader of the immunosensing device.
In one embodiment, the bulk high-field permanent magnet is
substantially cylindrical, having a diameter in the range of about
0.1 mm to about 5 mm and a length of about 0.1 mm to about 5 mm,
and is positioned to yield an "event horizon" (as defined herein)
in the conduit suitable for bead capture within a short period of
time (e.g., 1-5 minutes). The conduit generally has a height of
about 0.2 mm to about 5 mm and a width of about 0.2 mm to about 5
mm, and either a uniform or non-uniform cross-sectional area. In
other embodiments, the bulk magnet shape may be in the form of a
square, rectangle, oval, flake, pyramid, sphere, sub-sphere, or
other shaped form.
The method of some embodiments of the invention includes (a) mixing
magnetically susceptible beads coated with a capture antibody with
a sample suspected of containing an analyte, and a signal antibody
to form a sandwich on the beads, (b) applying the mixture to the
immunosensor and magnetically localizing and retaining at least a
portion of the beads on the immunosensor, (c) washing the unbound
sample from the immunosensor, and (d) exposing the signal antibody
of the sandwich to a signal generating reagent, and measuring a
signal from the reagent at the electrode. In some embodiments, the
method can use magnetically susceptible beads and signal antibodies
dissolved from a dry matrix. In other embodiments, the method
operationally relies on step (a) occurring in a first portion of a
conduit and step (b) occurring in a second portion of a conduit
where the sensor is located.
2. Magnetized Layer Integral to Magnetic Immunosensing Device
Another embodiment of the present invention includes a magnetic
immunosensing device, which comprises a sensing electrode on a
substantially planar chip. The electrode is positioned in a conduit
for receiving a sample mixed with antibody-labeled magnetically
susceptible beads and a magnetized layer (e.g., microfabricated
magnetic layer). The magnetized layer may be included on (e.g.,
positioned over, directly attached, coated or patterned onto any
surface of the chip) or embedded into the chip (e.g., positioned
within the chip, integral to the chip). This configuration attracts
the magnetically susceptible beads substantially proximate to the
electrode and substantially retains them at the electrode during
removal of unbound sample and washing of the electrode.
The magnetized layer preferably is formed from a mobile magnetic
composition, e.g., a slurry, comprising a material capable of
sustaining a high-field permanent magnetic field, e.g., a NdFeB
alloy, as particles in an immobilization or support matrix (e.g., a
polyimide, polyvinyl alcohol (PVA) or thermoplastic equivalent).
This slurry is not limited by viscosity and can include any
viscosity suitable for application. In various optional
embodiments, the mobile magnetic composition has a viscosity
ranging from 0.3 to 300,000 CPS, e.g., from 100 to 100,000 CPS or
from 1,000 to 10,000 CPS. The magnetic particles in the slurry of
certain embodiments of the invention have an average particle size
from 0.01 .mu.m to 100 .mu.m, e.g., from 0.1 .mu.m to 10 .mu.m or
from 3 .mu.m to 7 .mu.m.
In addition to polyimide, PVA and thermoplastic polyimide, two-part
chemically cured epoxy resins, kapton and the like may be used as
the support matrix for fixing the magnetic particles to the wafer.
The methods of curing the matrix may be based on a photo-initiated,
thermally initiated or chemically initiated process. In certain
embodiments, the immobilization matrix is comprised of other
photoformed matrix materials.
As provided above, the slurry can be applied in a variety of
locations in or on the immunosensing device (e.g., to the front
side or backside of a wafer or chip, electrode, housing, reader,
etc.). For example, in some embodiments of the invention, the
high-field permanent magnetic material is applied to the
substantially planar chip in a patterned manner (e.g., using a
mask). In certain embodiments, the high-field permanent magnetic
material is also applied to a microfabricated sensing electrode. In
other embodiments, the slurry is applied in a layer below the
sensing electrode.
Prior to the application process, the slurry may or may not be
magnetized. However, after the deposition step, the magnetic layer
preferably is magnetized to provide directionality to the
field.
B. Sensing Electrode
The sensing electrode is preferably microfabricated (e.g., an
amperometric gold array) on a substantially planar chip (e.g.,
silicon wafer), as described in the jointly-owned pending and
issued patents cited herein (e.g., U.S. Pat. Nos. 5,200,051 and
7,419,821).
C. Magnetically Susceptible Beads
In various embodiments of the invention, the biological sample,
e.g., blood sample, is amended with magnetically susceptible beads.
The magnetically susceptible beads may be comprised of any material
known in the art that is susceptive to movement by a magnet (e.g.,
permanent magnet or electromagnet) utilized in or in concert with
the device of the present invention. As such, the terms "magnetic"
and "magnetically susceptible" with regard to beads can be used
interchangeably.
In some embodiments of the invention, the beads include a magnetic
core, which preferably is completely or partially coated with a
coating material. The magnetic core may comprise a ferromagnetic,
paramagnetic or a superparamagnetic material. In preferred
embodiments, the magnetically susceptible beads comprise a ferrite
core and an outer polymer coating. However, the magnetic core may
comprise one or more of Fe, Co, Mn, Ni, metals comprising one or
more of these elements, ordered alloys of these elements, crystals
comprised of these elements, magnetic oxide structures, such as
ferrites, and combinations thereof. In other embodiments, the
magnetic core may be comprised of magnetite (Fe.sub.3O.sub.4),
maghemite (.gamma.-Fe.sub.2O.sub.3), or divalent metal-ferrites
provided by the formula Me.sub.1xOFe.sub.3+xO.sub.3 where Me is,
for example, Cu, Fe, Ni, Co, Mn, Mg, or Zn or combinations of these
materials, and where x ranges from 0.01 to 99.
Suitable materials for the coating include synthetic and biological
polymers, copolymers and polymer blends, and inorganic materials.
Polymer materials may include various combinations of polymers of
acrylates, siloxanes, styrenes, acetates, akylene glycols,
alkylenes, alkylene oxides, parylenes, lactic acid, and glycolic
acid. Biopolymer materials include starch or similar carbohydrate.
Inorganic coating materials may include any combination of a metal,
a metal alloy, and a ceramic. Examples of ceramic materials may
include hydroxyapatite, silicon carbide, carboxylate, sulfonate,
phosphate, ferrite, phosphonate, and oxides of Group IV elements of
the Periodic Table of Elements.
In other embodiments of the invention, the magnetic beads comprise
non-magnetic substrate beads formed, for example, of a material
selected from the group consisting of polystyrene, polyacrylic acid
and dextran, upon which a magnetic coating is placed.
In principal, any correctly-sized magnetically susceptible bead
capable of being positioned with the high-field magnet of the
present invention may be utilized, taking into account the
dispersability requirements for the magnetically susceptible beads.
In preferred embodiments, at least 50 wt. %, e.g., at least 75 wt.
%, of the magnetically susceptible beads are retained at the
electrode surface. In some exemplary embodiments, the average
particle size of the magnetically susceptible beads may range from
0.01 .mu.m to 20 .mu.m, e.g., from 0.1 .mu.m to 10 .mu.m, from 0.1
.mu.m to 5 .mu.m or from 0.2 .mu.m to 1.5 .mu.m. As used herein,
the term "average particle size" refers to the average longest
dimension of the particles, e.g., beads, for example the diameter
for spherical particles, as determined by methods well-known in the
art. The particle size distribution of the magnetically susceptible
beads preferably is unimodal, although polymodal distributions may
also be used in accordance with the present invention. While use of
a spherical magnetically susceptible bead is preferred, in other
embodiments, other bead shapes and structures, e.g., ovals,
sub-spherical, cylindrical and other irregular shaped particles,
are within the meaning of the term "beads" and "microparticles" as
used herein.
Commercial sources for magnetically susceptible bead preparations
include Invitrogen.TM. (Carlsbad, Calif., U.S.A.) by Life
Technologies.TM., Ademtech (Pessac, France), Chemicell GmbH
(Berlin, Germany), Bangs Laboratories, Inc..TM. (Fishers, Ind.) and
Seradyn, Inc. (Indianapolis, Ind.). Many of the commercially
available products incorporate surface functionalization that can
be employed to immobilize antibodies (e.g., IgG) on the bead
surfaces. Exemplary functionalizations include carboxyl, amino or
streptavidin-modified magnetically susceptible beads.
The magnetically susceptible beads are preferably coated with an
antibody to an analyte that is a cardiovascular marker, e.g.,
cardiac troponin I, troponin T, a troponin complex, human chorionic
gonadotropin, BNP, creatine kinase, creatine kinase subunit M,
creatine kinase subunit B, creatine kinase MB (CK-MB), proBNP,
NT-proBNP, myoglobin, myosin light chain or modified fragments
thereof, among others. In addition, markers for other indications
can be utilized. Further exemplary analytes include, but are not
limited to, beta-HCG, TSH, ultra hTSH II, TT3, TT4, FT3, FT4,
myeloperoxidase, D-dimer, CRP, NGAL, PSA, LH, FSH, galectin-3,
prolactin, progesterone, estradiol, DHEA-S, AFP, CA 125 II, CA 125,
CA 15-3, CA 19-9, CA 19-9XR, CEA, thyroxine (T4), triiodothyronine
(T3), T-uptake, Tg, anti-Tg, anti-TPO, ferritin, cortisol, insulin,
HBsAg, HCV Ag/Ab combo, HCV core Ag, anti-HCV, AUSAB (anti-HBs),
CORE, CORE-M, SHBG, iPTH, theophylline, sirolimus, tacrolimus,
anti-HAV, anti-HAV IgM, HAVAB, HAVAB-M, HAVAB-M2.0, HAVAB-G, HAVAB
2.0, HAVAB 2.0 Quant, IgM, CMV IgM, CMV IgG, a-2-microglobulin,
digitoxin, HBe, anti-HBe, HBeAg, HIV 1/2gO, HIV Ag/Ab combo,
testosterone, SCC, vitamin B12, folate, syphilis, anti-HBc, rubella
IgG, rubella IgM, homocysteine, MPO, cytomegalovirus (CMV) IgG
Avidity, toxo IgG avidity, toxo IgG, toxo IgM, C-peptide, vitamin
D, HTLV I/II, total ahCG, progesterone, estradrogen, prolactin,
myoglobin, tPSA, fPSA, carbamazepine (CBZ), digoxin, gentamicin,
NAPA, phenytoin, phenobarbital, valproic acid, vancomycin,
procaine, quinidine, tobramycin, methamphetamine (METH),
amphetamine (AMPH), barbituates, benzodiazepine, cannabis, cocaine,
methadone, opiates, PCP, acetaminophen, ethanol, salicylates,
tricyclics, holoTc, anti-CCP, HbA1c, barbs-U, among others. In
certain embodiments of the invention, the antibody is to a
low-abundance analyte in the sample. Abbreviated names above will
be familiar to one of ordinary skill in the clinical analytical
art.
The magnetic immunosensor and methods of the present invention
preferably also comprise a second antibody, which is a labeled
antibody, also referred to herein as a signal antibody. In some
embodiments, the labeled antibody is in the form of a dissolvable
dry reagent, which also may comprise the magnetically susceptible
beads that are employed in the present invention, as discussed
below. Both the immobilized and labeled antibodies can be
monoclonal, polyclonal, fragments thereof and combinations thereof.
In addition, one or more of the antibodies can be labeled with
various labels including a radiolabel, enzyme, chromophore,
flurophore, chemiluminescent species, ionophore, electroactive
species and others known in the immunoassay art. Where the second
antibody is labeled with an enzyme, it is preferably ALP,
horseradish peroxidase, or glucose oxidase. In other embodiments,
the analyte is labeled with fluorescein, ferrocene, p-aminophenol,
or derivatives thereof.
In certain embodiments, the magnetically susceptible beads are
deposited in a suitable region of the magnetic immunosensing device
as a suspension in, for example, a mixture of lactitol and
DEAE-dextran such as that supplied by Advanced Enzyme Technologies
(Pontypool, Great Britain). Evaporation of the solvent, usually
water, yields a glassy deposit in which the beads are immobilized.
The lactitol/DEAE-dextran allows the beads to be regionalized
within the device in a mechanically and biochemically stable state,
but which also rapidly dissolves upon contact with a sample.
In other embodiments, the magnetically susceptible beads are
homogeneously mixed with the sample. In still other embodiments,
the magnetically susceptible beads may be less homogeneously mixed
with the sample; however, the intent is to optimize the position
and concentration of the beads relative to the sensing electrode.
Those skilled in the art will recognize that the magnetically
susceptible beads of the present invention may be added to the
biological sample prior to introduction into the magnetic
immunosensing device, such as, for example, as an integral part of
a blood collection device or as a standard manual addition step.
However, for the convenience of the user and to assure a quality
assay, the magnetically susceptible beads are preferably included
within the device.
In some embodiments of the invention, the sample, e.g., whole blood
sample, is collected and then amended by dissolving a dry reagent
comprising the magnetically susceptible beads into the sample. Any
portion of the immunosensing device may be coated with the dry
reagent (e.g., sensing device, conduit, sample entry port, sample
holding chamber). In addition to the magnetically susceptible
beads, the dry reagent may further include one or more of: beads
for reducing leukocyte interference, a leukocidal reagent, buffer,
salt, surfactant, stabilizing agent, simple carbohydrate, complex
carbohydrate and various combinations thereof. The dry reagent can
also include an enzyme-labeled antibody (e.g., the above-described
labeled antibody) to the analyte.
In various embodiments, the magnetically susceptible beads are used
to amend the biological sample, e.g., blood, in a first container
or location, and then the sample is passed to a second container or
location that includes the capture and signal antibodies. In some
embodiments, the magnetically susceptible beads are contained in
solution and mixed with the biological sample, and the resulting
amended sample is introduced into the magnetic immunosensing
device. For example, a blood sample may be mixed with the
magnetically susceptible beads to form an amended sample, which is
then introduced into the device. In certain embodiments, the
magnetic immunosensing device, e.g., cartridge, includes a pouch
that contains a liquid comprising the magnetically susceptible
beads, which may be mixed with a biological sample in the device
and then processed substantially as described herein to form an
assay (e.g., sandwich assay) for analyte detection.
In other embodiments, electrowetting is employed to mix a first
liquid comprising the magnetically susceptible beads with a liquid
biological sample, e.g., blood. In one such embodiment, an
apparatus may be provided for manipulating droplets. The apparatus,
for example, may have a single-sided electrode design in which all
conductive elements are contained on one surface on which droplets
are manipulated. In other embodiments, an additional surface is
provided parallel with the first surface for the purpose of
containing the droplets to be manipulated. The droplets are
manipulated by performing electrowetting-based techniques in which
electrodes contained on or embedded in the first surface are
sequentially energized and de-energized in a controlled manner. The
apparatus may allow for a number of droplet manipulation processes,
including merging and mixing two droplets together, splitting a
droplet into two or more droplets, sampling a continuous liquid
flow by forming from the flow individually controllable droplets,
and iterative binary or digital mixing of droplets to obtain a
desired mixing ratio.
In addition, any immunoassay format known in the art may be
modified to include the magnetically susceptible beads of the
present invention, for example, by adding the beads in a sample
pre-treatment step. The pretreatment may be accomplished, for
example, by incorporating the beads in a blood collection device,
in a separate vessel, or may take place in the immunoassay device
itself by incorporation of the beads in the test cycle of the
device.
In various embodiments of the invention, the beads are mobile and
thereby capable of interacting with an analyte. After binding to
the analyte of interest, magnetic forces are used to concentrate
the beads at the electrode for measurement causing the magnetically
susceptible beads to be localized to the amperometric electrode for
signal detection. One advantage of using mobile beads according to
the present invention is that their motion in the sample or fluid
accelerates binding reactions, making the capture step of the assay
faster.
D. Additives
In some embodiments of the invention, additives may be included in
the magnetic immunosensing device or used in conjunction with the
assay. In certain embodiments, an anticoagulant can be added. For
example, heparin may be added to improve performance in cases where
the sample was not collected in a heparinized tube or was not
properly mixed in a heparinized tube. A sufficient amount of
heparin may be added so that fresh unheparinized blood will remain
uncoagulated during the assay cycle of the cartridge, typically in
the range of 2 to 20 minutes. In still other embodiments, one or
more of proclin, DEAE-dextran, tris buffer, and lactitol can be
added as reagent stabilizers. In further embodiments, a surfactant
such as polysorbate 20, also known as Tween.RTM. 20, can be added
to reduce binding of proteins to plastic, which is a preferred
material for the cartridge housing of the magnetic immunosensing
device. The addition of a surfactant also facilitates the even
coating of reagents on plastic surfaces and minimizes the
crystallization of sugars (e.g., lactitol). In other embodiments of
the invention, an antibacterial agent or biocide (e.g., sodium
azide) may be added to inhibit bacterial growth.
II. Manufacture of Magnetic Immunosensing Device
In one embodiment of the invention, a silicon wafer is thermally
oxidized to form an insulating oxide layer having a thickness of
about 1 .mu.m. A titanium/tungsten layer is then sputtered onto the
oxide layer to a preferable thickness of about 100 .ANG. to about
1000 .ANG., followed by a layer of gold that is from 500 .ANG. to
1000 .ANG. thick, most preferably about 800 .ANG. thick. Next, a
photoresist is spun onto the wafer and is dried and baked. The
surface is then exposed using a contact mask, the latent image is
developed, and the wafer is exposed to a gold-etchant. The
patterned gold layer is coated with a photodefinable polyimide,
suitably baked, exposed using a contact mask, developed, cleaned in
an oxygen plasma, and preferably imidized at 350.degree. C. for
about 5 hours. This leaves a large number of electrode openings in
the polyimide layer in a square array. In some embodiments, the
square array has a diameter, for example, from 2 .mu.m to 100
.mu.m, from 5 .mu.m to 15 .mu.m or about 7 .mu.m, with an
inter-distance of, for example, from 5 .mu.m to 100 .mu.m, from 10
.mu.m to 20 .mu.m or about 15 .mu.m. The area covered by these
electrodes (i.e., sensor area) is substantially circular with a
diameter of, for example, from 50 .mu.m to 1000 .mu.m, from 100
.mu.m to 300 .mu.m or about 300 .mu.m.
After dicing the wafer into individual chips, each chip is
assembled into a single-use cartridge. The cartridges may be of the
type described in U.S. Pat. Nos. 7,419,821 to Davis et al. or
jointly-owned U.S. Patent Application No. 61/288,189, entitled
"Foldable Cartridge Housings for Sample Analysis," filed Dec. 18,
2009, the entireties of which are incorporated herein by reference.
In one embodiment, the sensor is positioned in a conduit for
receiving a sample, and a high-field magnet, e.g., permanent or
electromagnet, is positioned directly below the sensor, preferably
in the center region thereof. In another embodiment, a high-field
magnet can be positioned above the sensor region of the conduit.
These elements may be in a fixed position within the instrument
housing, or adapted to an actuator capable of moving in and out of
position with respect to the immunosensor and conduit. The one or
more high-field magnets can be used for attracting magnetically
susceptible beads in the conduit (e.g., substantially proximate to
the sensor) and retaining them in the region of the sensor during
removal of sample and washing of the sensor to remove unbound or
partially absorbed reagents. As described above, the magnetic beads
are coated with an antibody to an analyte that may be present in
the sample.
A. Trenches
In the immunosensor embodiment of the invention shown in FIG. 1,
additional features are depicted. In particular, the
cross-sectional representation of FIG. 1 shows an electrochemical
sensor patterned in suitable material (e.g., photoformable
polyimide) with trenches (or grooves) having width of from about 1
.mu.m to about 100 .mu.m, e.g., from 3 .mu.m to 50 .mu.m or from 10
.mu.m to 20 .mu.m, height or depth of from about 0.1 .mu.m to about
100 .mu.m, e.g., from 1 .mu.m to 50 .mu.m or from 5 .mu.m to 10
.mu.m, and length of about 1 .mu.m to about 5000 .mu.m, e.g., from
10 .mu.m to 1000 .mu.m or from 100 .mu.m to 500 .mu.m. In this
immunosensor design embodiment, the planar chip is positioned into
an analytical system capable of applying magnetic fields in either
direction in roughly the z axis, as shown by vectors B1 and B2. In
some embodiments and as shown in FIG. 1, the trenches may be
oriented in a direction substantially parallel to the direction of
sample flow in the immunosensing device. In alternative
embodiments, the trenches may be oriented in a direction
substantially transverse to or perpendicular to the direction of
sample flow. The trench structure of the present invention may
beneficially inhibit fluid motion in the x and/or y directions from
removing any magnetically susceptible beads that have been
magnetically localized within a trench, for example, by a washing
fluid.
A top view illustration of the electrochemical sensor of FIG. 1 is
illustrated in FIG. 2, in which microelectrodes of diameter .mu.D
(i.e., about 1 .mu.m to about 100 .mu.m) and spacing .mu.L (about 5
.mu.m to about 500 .mu.m) are observed at the bottom of each groove
in the sensor structure as an array. These microelectrodes can be
comprised of gold or other suitable conductor and can be patterned
as described above (e.g., with gold and polyimide). In preferred
embodiments, the high-field magnet is positioned on the underside
or below the electrochemical sensor.
B. Magnetic Fields
In some embodiments of the invention, a sandwich immunoassay is
formed on the magnetically susceptible beads substantially
everywhere in the conduit and the beads are slowly collected on or
adjacent to a sensor surface by bringing the magnet proximal to the
cartridge and slowly oscillating the fluid. In certain embodiments,
the oscillating magnetic fields are produced by a moveable
high-field magnet, e.g., permanent magnet or electromagnet, located
inside the instrument. In other embodiments, one or more magnets
are stationary inside the instrument and the sandwich formation is
located in a position beyond the field. The sample may then be
directed, e.g., by a pump or similar means, into a region where the
magnetic field is sufficiently strong for capture.
In a preferred embodiment, the sample is oscillated in a back and
forth motion over the sensor, e.g., by one or more pumps, while the
magnetic field is applied, in order to maximize the opportunity for
the magnetically susceptible beads to be attracted to the sensor
surface.
III. Methods of Performing Immunoassays
The present invention is applicable to methods of performing
immunoassays with a magnetic immunosensor. In preferred
embodiments, the present invention may be employed in one or more
of the following areas: immunosensors, most notably in the context
of point-of-care testing; electrochemical immunoassays;
immunosensors in conjunction with immuno-reference sensors; whole
blood immunoassays; single-use cartridge based immunoassays; and
non-sequential immunoassays with only a single wash step; and dry
reagent coatings. As will be appreciated by those skilled in the
art, the general concept disclosed herein is applicable to many
immunoassay methods and platforms.
The methods of the invention are applicable to various biological
sample types (e.g., blood, plasma, serum, urine, interstitial fluid
and cerebrospinal fluid). The present invention is applicable a
variety of immunoassays including both sandwich and competitive
immunoassays.
A. Sandwich Immunoassay
In sandwich assay embodiments, the sample contacts the immunosensor
with an immobilized first antibody to the target analyte, and a
labeled second antibody to said target analyte. In some embodiments
of the invention, the sample, e.g., whole blood sample, is
collected and then amended by dissolving a dry reagent comprising
magnetically susceptible beads into the sample. As discussed above,
the beads preferably include an antibody to the analyte of interest
immobilized on the outer surface thereof. The dissolution of the
dry reagents and the sandwich formation step can occur concurrently
or in a stepwise manner.
The magnetically susceptible bead concentration employed may vary
widely. In some exemplary embodiments, the sample is amended with
the magnetically susceptible beads to provide a dissolved bead
concentration of at least 5 .mu.g per .mu.L of sample, e.g., at
least 10 .mu.g per .mu.L of sample, or at least 15 .mu.g per .mu.L
of sample. The dry reagent preferably dissolves into the sample to
give a bead concentration of from about 5 .mu.g to about 40 .mu.g
beads per .mu.L of sample, preferably from about 10 to about 20
.mu.g beads per .mu.L of sample. Depending on the size of the
beads, this corresponds to at least about 10.sup.4 beads per .mu.L
of sample, at least about 10.sup.5 beads per .mu.L of sample, or
approximately from about 10.sup.5 to about 10.sup.6 beads per .mu.L
of sample. Thus, in some preferred embodiments, the beads are
present in an amount sufficient to provide a dissolved bead
concentration of at least 10.sup.4 beads per .mu.L of sample, e.g.,
at least about 10.sup.5 beads per .mu.L of sample, or from about
10.sup.5 to about 10.sup.6 beads per .mu.L of sample. Once this
step is completed, it is possible to perform an immunoassay, e.g.,
an electrochemical immunoassay, on the amended sample to determine
the concentration of an analyte. In preferred embodiments of the
invention, at least about 10,000 beads are used for each assay.
This lower limit reduces counting error issues, e.g., about 1% or
greater for about 1,000 beads or less. Defining an upper limit is
less straightforward and depends on bead size, however about
10,000,000 beads is generally sufficient. In certain embodiments,
the dissolved bead volume is less than about 1% of the total sample
assay volume, and is preferably less than about 0.1%.
In the actual assay step, in preferred embodiments, once the
sandwich is formed between the immobilized and signal antibodies on
the outer surface of the magnetically susceptible beads, a magnetic
field is applied to attract the beads in the conduit substantially
proximate to the electrode. The sample is subsequently washed to a
waste chamber while leaving the retained beads substantially
proximate to the electrode, followed by exposing the sandwich on
the magnetically susceptible beads to a substrate capable of
reacting with an enzyme to form a product capable of
electrochemical detection. One exemplary format is an
electrochemical enzyme-linked immunosorbent assay.
In some embodiments of the invention, a magnetized layer (e.g.,
microfabricated magnetic layer) can be used as the basis for a
sandwich immunoassay method. For example, in one embodiment, the
invention is to a method of performing an immunoassay with a
magnetic immunosensor where the immunosensor comprises a sensing
electrode on a substantially planar chip and an additional layer on
the chip that is magnetized, positioned in the region of the
electrode. This method comprises (a) mixing magnetically
susceptible beads coated with a capture antibody with a sample
containing (or suspected of containing) an analyte and a signal
antibody to form a sandwich on the beads, (b) applying the mixture
to the immunosensor and magnetically localizing and retaining at
least a portion of the beads on the immunosensor, (c) washing the
sample and unbound material from the immunosensor and exposing the
signal antibody of the sandwich to a signal generating reagent, and
(d) measuring a signal from the reagent at the electrode.
Furthermore, step (a) preferably occurs in a first portion of a
conduit and step (b) preferably occurs in a second portion of a
conduit where the sensor resides. In some embodiments, other
features of the assay (e.g., sample type, the use of dry reagents
including magnetically susceptible beads and the signal antibodies,
as described herein) can be optimized to facilitate the dissolution
of a dry matrix into the sample.
B. Competitive Immunoassay
Embodiments of the present invention are also applicable to methods
of performing a competitive immunoassay with a magnetic
immunosensor. In traditional competitive assay embodiments, a
sample contacts an immunosensor comprising an immobilized first
antibody to a target analyte, and a labeled target analyte that
competes for binding with the target analyte. Because the
(unlabeled) target analyte competes with (amended) labeled target
analyte, the resulting signal is inversely proportional to the
native analyte concentration of the sample.
In some such embodiments, the method of the invention includes
mixing magnetically susceptible beads coated with a capture
antibody with a sample containing (or suspected of containing) an
analyte and an added labeled form of the analyte to permit
competitive binding on the magnetically susceptible beads, applying
the mixture to an immunosensor and magnetically localizing and
retaining at least a portion of the beads on the immunosensor,
washing the sample and unbound material from the immunosensor,
exposing the labeled analyte to a signal generating reagent, and
measuring a signal from the reagent at the sensing electrode.
C. i-STAT.RTM. Immunoassay
While the present invention is broadly applicable to immunoassay
systems, it is best understood in the context of the i-STAT.RTM.
immunoassay system (Abbott Point of Care Inc., Princeton, N.J.,
USA), as described in the jointly-owned pending and issued patents
cited herein.
In these immunoassay systems, only a small fraction of analyte
present in the sample is captured. The assay involves sampling of
an analyte present in plasma or whole blood by capturing analyte on
an antibody-labeled microparticle which is coated (or permanently
bound) to the surface of a sensor, e.g. an electrochemical sensor.
In certain embodiments, a second antibody labeled with an enzyme
then binds to the analyte to make a sandwich. The electrochemical
detection format ensures that substantially all the enzyme (e.g.,
alkaline phosphatase (ALP)) is detected (i.e., detection efficiency
approaches 100%), which allows for a high sensitivity assay.
Typically, however, the overall amount of analyte that is in the
region of the antibody-labeled microparticle and susceptible to
capture during normal usage is low due to mass transport
limitations. It would be advantageous, therefore, to capture a
higher percentage of the analyte, while still retaining the high
efficiency of the detection step. As disclosed in the present
invention, more analyte advantageously may be captured and detected
by employing capture of a magnetically susceptible bead reagent
that is distributed (e.g., homogeneously distributed) throughout
the sample during the analyte capture step, but is magnetically
localized to the sensor in such a way that retains the ability to
measure the enzyme with sufficiently improved detection
efficiency.
In certain embodiments, the sample, e.g., plasma or whole blood
sample, is amended with interference-reducing and/or conditioning
reagents optionally located in a sample inlet print of a cartridge.
For example, the reagents may contain immunoglobulins,
immunoglobulin-coated non-magnetically susceptible beads,
non-magnetically susceptible bead reagents for interference
screening, and other stabilizing or conditioning reagents. See,
e.g., U.S. patent application Ser. Nos. 12/620,230 and 12/620,179,
both filed Nov. 17, 2009, each of which is incorporated herein by
reference in its entirety, for a description of the use of
sacrificial beads for reducing or eliminating interference caused
by leukocytes in a blood sample. See also U.S. patent application
Ser. No. 12/411,325, the entirety of which is incorporated herein
by reference, which describes the use of non-human IgM and/or IgG
or fragments thereof to reduce interference caused by heterophile
antibodies.
Upon being pushed into a conduit, the sample is amended with at
least two reagents, a primary capture reagent and a labeled
conjugate reagent. The primary capture reagent comprises
magnetically susceptible beads coated with antibodies or antibody
fragments appropriate to the analyte of interest. This magnetically
susceptible bead reagent may be printed, for example, on a wall
portion of the holding chamber or attached to a conduit in a region
upstream of the sensor chip, using methods described in
jointly-owned U.S. Pat. No. 5,554,339 to Cozzette et al., which is
hereby incorporated by reference in its entirety. The labeled
conjugate reagent preferably also comprises a signaling element
(e.g., ALP). This reagent may be printed, for example, as a
dissolvable matrix with the primary capture reagent in a one stage
assay. In another embodiment, the two reagents are amended into the
sample separately from one another, in either order.
To promote sandwich formation on the magnetically susceptible
beads, it is desirable that the amended sample be oscillated or
mixed in the conduit for an appropriate period of time (e.g., about
1 minute to about 20 minutes or about 5 to 10 minutes). This allows
analyte present in the sample to be captured on the surface of the
beads and labeled with a signal-generating conjugate.
In one embodiment, following capture, magnetic field B1 is applied
in such a way that the magnetically susceptible beads are induced
to migrate to the top of the sensor channel, opposite the sensor
surface, where they are temporarily retained. The surface (i.e.,
top of sensor channel) may be patterned in such a way that there is
a tendency of the beads to resist (beyond the action of B1)
movement in the direction of mixing upon subsequent fluid movement.
This process can be accompanied by low-amplitude oscillations in
the direction of the capture motion (x-axis) in order to assist
capture of the magnetically susceptible beads without entrapping
formed elements or non-magnetically susceptible beads.
In one embodiment, the sample is then moved to a waste chamber.
However, in a preferred embodiment, the sample is moved to a
lock-wick feature by means of pressurization actuated by an
air-bladder as described in jointly-owned U.S. Pat. No. 7,419,821
to Davis et al. (referenced above) and U.S. Pat. No. 7,723,099 to
Miller et al. (referenced above). Each of these patents is hereby
incorporated by reference in its entirety.
The movement of the magnetically susceptible beads is conducted in
such a way that the magnetic field B1 is able to temporarily retain
the beads at the ceiling of the sensor channel, preferably
substantially opposite the sensor. During this period, the sensor
channel is washed in a fashion similar to that described in U.S.
Pat. Nos. 7,419,821 and 7,723,099 (referenced above). In preferred
embodiments, prior to completion of the washing step, a portion of
the wash fluid is left in the sensor channel until completion of
the following magnetic actuation step. In this embodiment, the
magnetic field B1 is reversed to B2 with the effect that the beads
formerly held in position at the sensor channel ceiling now migrate
towards the trenches or grooved sensor structure. Low amplitude
oscillations of the fluid portion in the direction of capture
mixing can be applied in order to help settle the beads into the
trenches of the immunosensor. Upon completion of this step, the
wash fluid is slowly pulled from the sensor channel and optionally
the magnetic field B2 is turned off. A current arising from the
diffusion of an electroactive species generated by the action of
bound label on a suitable electrogenic substrate contained in the
wash fluid is then measured. (See, for example, U.S. Pat. Nos.
7,419,821 and 7,723,099 (referenced above)).
As described above, the trenches or grooved structure of the
immunosensor are intended to aid capture of the magnetically
susceptible beads and yet allow for efficient washing in the
direction of fluid movement. However, other suitable structures may
also be used. One exemplary structure includes a grid (e.g.,
rectilinear) array. In this embodiment, the ability to retain the
beads over the array will generally depend on the ability to focus
magnetic fields in such a way that highest field densities are
contained within the area demarcated by the area of the array.
In some embodiments, it is desirable to seek to capture
substantially all or a reliable fraction (e.g., over 75 wt. %) of
the beads, in which case the dimensions of the bead retention
feature (e.g., trenches) are of secondary importance provided that
they supply sufficient volume to contain the beads. Alternatively,
a fixed proportion of the beads may be sampled in which case the
total volume of the capture feature(s) must be held constant.
Certain embodiments of the invention utilize sequential application
of two opposing magnetic fields. In other embodiments, a single
applied field may be utilized. Furthermore, in some embodiments,
the ability to provide sufficient substrate to the enzyme-limited
detection reaction may require high substrate concentrations (e.g.,
about 20 mM).
The number and dimensions of the optional trenches or other
retention features are dependent on the size and number of the
magnetically susceptible beads required to achieve efficient
capture of analyte present in the sample. In some embodiments, the
length of the trenches, for example, may be several thousand
microns while the height and width may be on the order of several
microns. In addition, one function of the trenches is to allow for
localization and consolidation of the beads and enhanced resistance
against fluid motion to dislodge them. In certain embodiments, the
beads inside the trenches are mobile.
In other embodiments, after dicing the wafer into individual chips,
each chip preferably is assembled into a single-use cartridge and
in this example a standard immunosensor is used without the groove
features. The sensor is positioned in a conduit for receiving a
sample, wherein a single high-field magnet, e.g., permanent magnet
or electromagnet, preferably a neodymium iron boron magnet (e.g.,
Nd.sub.2Fe.sub.14B), is also positioned directly below the center
of the sensor. The high-field magnet is used for attracting
magnetically susceptible beads in the conduit (e.g., substantially
proximate to the sensor) and retaining them in the region of the
sensor during removal of sample and washing of the sensor. In
preferred embodiments, the beads are coated with an antibody to an
analyte in the sample or suspected of being present in the
sample.
FIGS. 3A-C shows three exemplary configurations of the sensor chip
or die in a base or cartridge housing of the type described in
jointly-owned U.S. Pat. Nos. 7,419,821 and 7,723,099 (referenced
above) or U.S. Patent Application No. 61/288,189 (referenced
above). The oval structure on the sensor die corresponds to the
immunosensor, which is positioned in the conduit. In each
embodiment, the high-field permanent magnets are cylindrical with
lengths of from 1 mm to 10 mm, e.g., from 2 mm to 5 mm, preferably
about 3 mm, and diameters of from 0.1 mm to 5 mm, e.g., from 0.5 mm
to 2 mm. In FIGS. 3A-C, the magnets have diameters of about 1 mm,
about 0.5 mm and about 0.3 mm, respectively. The magnets are
abutted to the underside of the chip, which preferably has a
thickness of from about 0.2 mm to 5 mm, e.g., from 0.5 mm to 2 mm
or preferably about 1 mm. FIG. 4 is a schematic representation of
the magnetic field lines as the magnet diameter decreases from 1 mm
to 0.3 mm and illustrating how the field and magnet selection may
impact where on the sensor the magnetically susceptible beads are
attracted and focused.
FIG. 5 is a micrograph of magnetically susceptible beads captured
on a chip surface where the center of the magnet having a diameter
1 mm is positioned directly below a point on the perimeter of an
immunosensor. This configuration assisted with showing the contrast
between beads (black), the gold electrode area (white) and the base
silicon material (gray). As shown in FIG. 5, a majority of the
beads are localized onto the area of the surface directly above the
magnet when a suspension of the beads is passed down the conduit
and into the region of the magnet for capture. The beads were about
3 .mu.m in diameter.
An immunoassay for cardiac troponin I (cTnI) was performed with a
0.5 mm diameter high-field permanent magnet positioned directly
under the center of an immunosensor. Unlike the embodiments using
two magnets described above, in this embodiment, the bead sandwich
formation step is performed in a portion of the conduit upstream
from the sensor, so as to avoid localizing the beads onto the
sensor prematurely. FIG. 6 includes three traces of the
electrochemical detection step (chronoamperometry) for the
magnetically susceptible bead capture assay: plasma with zero cTnI;
Cliniqa control level 3; and Cliniqa control level 4. These bitmap
curves were run in substantially the same way as a commercial cTnI
cartridge, but with special software for the new fluidic motions.
All the reagents for this particular experiment were printed on the
sensor chip. The cTnI levels L3 and L4 were about 11 ng/mL and 40
ng/mL respectively. Each trace shows the current at the electrode
as a function of time. The rise time of the current to a
steady-state value reflects the time constant (TC) for the sensor
and the plateau value reflects the amount of analyte in the sample.
The traces in FIG. 6 show only the detection step, the complete
assay took about 12 minutes, which is acceptable for quantitative
point of care immunoassays.
Embodiments of the present invention demonstrate that using a fixed
permanent magnet in the present device can reliably capture beads
(e.g., superparamagnetic or ferromagnetic beads) onto an
immunosensor without substantial agglomeration of the beads. In
addition, the cartridge fluidic system disclosed in jointly-owned
U.S. Pat. Nos. 7,419,821 and 7,723,099 (referenced above) or U.S.
Patent Application No. 61/288,189 (referenced above) provide a
foundation for controlling the sample so that it is amended with
reagents and allowed time to react before it passes through to the
region of the conduit with the magnet. FIG. 7 illustrates the
positioning of a rare earth permanent magnet R21 below an
immunosensor chip within a cartridge housing in accordance with one
embodiment of the present invention, and FIG. 8 illustrates a
foldable cartridge of the type described in previously referenced
U.S. Patent Application No. 61/288,189 (referenced above), where
the magnet, e.g., rare earth permanent magnet (not shown), may be
positioned underneath immunosensor chip 204 or one of the other
adjacent chips.
In some embodiments, an immunosensor is provided where the magnetic
component is directly integrated into sensor manufacture, rather
than being a separate component (e.g., bulk permanent high-field
magnet) requiring assembly into the test device. For example, in
one embodiment, a mixture of photoformable polyvinyl alcohol (PVA)
mixed with ground Nd.sub.2Fe.sub.14B powder was printed onto a
wafer using a microdispensing apparatus of the type described in
jointly-owned U.S. Pat. No. 5,554,339 (referenced above). The
printed area was of a diameter of about 400 .mu.m. After exposure
to UV light and a wash step, the adhered layer was exposed to a
solution of magnetically susceptible beads of about 3 .mu.m
diameter. The solution was then removed and the surface washed with
buffer. FIG. 9 is a micrograph of a portion of the printed area
where the relatively smaller ferromagnetic (dark) beads are
accurately localized on the patterned magnetic layer. As shown,
small areas of the relatively larger and irregular NdFeB (light)
particles are observable below the beads. FIG. 10 is a micrograph
of a fractured cross-section of the device in FIG. 9.
FIGS. 11A-C illustrate details of the patterned PVA and polyimide
films with various particle sizes of NdFeB (FIG. 10A); ground 6
.mu.m Magnaquench particles (MQP) in polyimide (FIG. 10B); and
ground 6 .mu.m MQP in polyimide (FIG. 10C), before exposure to the
magnetically susceptible beads. These types of layers were formed
using Magnaquench particles (MQP), which is a NdFeB powder with an
average particle size of about 6 .mu.m in polyimide or Shipley
Photo Resist (SPR).
It was found in accordance with certain embodiments of the
invention that the about 3 .mu.m to about 7 .mu.m thick magnetic
film of FIG. 9 was partially less effective in terms of capturing
the beads than the about 30 .mu.m to about 40 .mu.m thick films of
FIGS. 11A-C. While the "capture radius" of the embodiment of FIG. 9
was several tens of microns, the capture radius of the embodiment
of FIGS. 11A-C was at least 200 .mu.m. In preferred embodiments,
the average capture radius of the sensors is less than about 500
.mu.m, e.g., less than 300 .mu.m or less than 250 .mu.m. The shape
and position of the magnet as well as its composition aid in the
sensitivity and precision of assays using the techniques of the
present invention.
FIG. 12A is a micrograph of a MQP NdFeB powder with an average
particle size of about 6 .mu.m. FIG. 12B is a micrograph of the MQP
NdFeB powder of FIG. 12A that has been comminuted via ball milling
for about 3 days. In some embodiments, the communited NdFeB powder
provides for a more homogeneous mobile magnetic composition, which
may facilitate magnetic capture of the beads. FIG. 13 is a
micrograph of beads captured on NdFeB particle surfaces. Those
skilled in the art will recognize that caution should be taken
during the grinding process to prevent combustion, e.g., adding 0.1
wt.% sulphur or cooling prior to opening the grinding
container.
FIGS. 14A and 14B show exemplary base immunosensor electrode arrays
110 partially covered with a printed polyimide and NdFeB particle
matrix 111 leaving a portion of the perimeter of the array exposed.
Also shown is the base silicon wafer 112 and a conductive line 113
for connecting the array to the instrument electronics. FIGS. 15A
and 15B depict the over-printed magnetic layer in accordance with
other embodiments of the present invention, where FIG. 15A is a
standard microelectrode array and FIG. 15B is a single ring
electrode having a width of about 20 .mu.m. FIG. 16 is a sheared
(i.e., fractured) sensor illustrating the printed magnetic layer
profile of FIGS. 15A and 15B.
It is preferable that the high-field magnet, e.g., permanent or
electromagnet, be within a few tens of microns of the amperometric
sensor electrode surface in order to speed up the time constant
(TC) of the sensor. In addition, it is preferable to size the
magnet so that the attraction of the magnetically susceptible beads
substantially dominates any of the potentially disruptive fluidic
steps (e.g., washing), thereby "effectively permanently" trapping
the reagent beads once captured on the immunosensor surface. While
not being bound by theory, this is equivalent to a concept of an
"event horizon" for the permanent magnet. "Effective permanent"
capture relates to the attraction of a magnetic particle to the
magnet, at the point at which the acceleration of the particle is
greater than any of the potentially "disruptive" fluid motions
(e.g., mixing oscillations and washing). Physical observation of
this phenomenon with a microscope, therefore, can give a practical
rough estimate of the "event horizon" for any given bead, sensor
design and fluidic motion. Such information is useful in refining
the overall test system design to achieve capture of substantially
all or a reliable fraction (e.g., over 75 wt. %) of the beads from
device to device. One intended use of the present invention is in
making single-use disposable test cartridges in large volumes
(i.e., greater than a million per year) and each device must
perform reproducibly within a given batch, (i.e., have clinically
acceptable precision and accuracy).
By way of example, in a preferred embodiment of a cTnI assay, a 10
.mu.L segment of blood sample is oscillated over the immunosensor
in a conduit of 0.5 mm height using a prototype design adapted from
the i-STAT.RTM. immunoassay cartridge format. This process is
performed with a 4 second cycle time (i.e., 4 Hertz) and equates to
a maximum fluid velocity of about 10 cm per second. The
magnetically susceptible beads are observed to move at about 10
.mu.m per second in the direction of the magnetic field
(microscope, jig and cartridge combination not shown). However,
once the beads are within about 50 .mu.m of the sensor surface, the
beads are within the "event horizon" and become captured on the
surface. The beads are no longer subject to the influence of the
oscillation (i.e., resuspension of the particle is substantially
negligible). Similarly, the trenched design of one embodiment of
the present invention (with the intentional fabrication of a
stagnant layer) may facilitate the retention of beads at the
surface during this step.
Unlike assays where magnetically susceptible beads are always in
contact with a liquid phase, in certain embodiments of the present
invention, one or more intervening air segments are present between
the sample and wash fluid in the conduit. (See, for example, U.S.
Pat. No. 7,419,821 (referenced above)). Surprisingly and
unexpectedly, it was found that the menisci formed by the air
segments provide a greater shear force to the captured beads as
they pass over the sensor than that generated by fluid oscillation
where the beads are in constant contact with the liquid phase. It
was also found that the slow passage of the menisci over the sensor
surface was more disruptive than a relatively faster motion. The
high-field magnets employed in the present invention (e.g., any
material that provides a high magnetic field (e.g., greater than
about 0.1 Tesla)) are preferable for optimizing movement of the
magnetically susceptible beads in relation to the sensor.
Those skilled in the art will recognize that consistently and
reliably retaining the captured beads on the sensor during the wash
step is desirable in the delivery of accurate analytical results.
While the addition of trenches on the sensor may be advantageous,
selection of the appropriate field strength was also found to be a
significant parameter. Experiments showed that greater than about
75% of the beads were retained on the surface during the wash step
of the methods of the present invention. In one exemplary
embodiment, the wash fluid comprises a 0.1 M diethanolamine buffer
(pH 9.8), 1 mM MgCl.sub.2, 1.0 M NaCl, 10 mM 4-aminophenylphosphate
and 10 .mu.M NaI.
With regard to the optimized test system design, the dimensions of
the high-field magnet are important in that, generally, if the
magnet is too small, either the time needed to capture the
magnetically susceptible beads is too long or the stability (i.e.,
retained capture) during the washing step will degrade the assay
performance. In addition, if the magnet is too deep below the
sensor plane, or held too far away from the sensor surface, the
force of attraction will be reduced and more diffuse and the
magnetic reagents are poorly focused upon capture. Based on the
present disclosure, those skilled in the art will understand how to
optimize these various requirements for any given system and
geometry (e.g., electrode area and position in a conduit).
In some immunosensor embodiments, where the bulk permanent
high-field magnet is positioned proximate to the sensing electrode
or sensor (e.g., in the housing of the magnetic immunosensing
device), the magnet diameter or width affects the time constant
(TC) of the sensor and how effectively the sensor detects the
signal-generating enzyme labels bound to the magnetic reagents on
the magnet surface. Taking this into account as well as the
relatively high topography designs shown in FIGS. 9 and 11A-C,
vis-a-vis the common desire to perform wafer fabrication processing
on substantially planar surfaces, the process illustrated in FIG.
17 was developed. FIG. 17 illustrates the etched trench process in
accordance with one embodiment of the present invention, wherein a
silicon wafer 1301 with a surface coating of photoresist 1302 is
etched first with hydrofluoric acid (HF) and then with hot
potassium hydroxide (KOH) or trimethyl ammonium hydroxide (TMAH) to
leave a trench of controlled dimensions 1303 (e.g., a depth and
width of from about 5 .mu.m to about 200 .mu.m). A slurry of
magnetizable particles (e.g., NdFeB powder) in a thermoplastic
matrix (e.g., polyimide) is then microdispensed 1305 or spin-coated
1306 into the trench thereby forming a substantially flat surface
co-planar with the wafer 1301. The wafer 1301 may be further
processed, as described in jointly-owned U.S. Pat. Nos. 7,419,821
and 7,723,099 (referenced above), to provide an immunosensor array
over each etched trench on a wafer.
FIG. 18 is a top view of an exemplary underside trench design
etched into a silicon wafer using a 800 .mu.m.times.1000 .mu.m
mask. FIG. 19 is the after etch cross-sectional profile of the
underside trench, having a depth of 50-90% into the silicon wafer.
FIGS. 20A and 20B depict different views of the etched trench in
accordance with one embodiment of the present invention and FIG. 21
depicts the trench filled with NbFeB powder in a polyimide resin.
FIGS. 22A and 22B are micrographs of a rectangular trench produced
on a silicon substrate via reactive ion etching by INO (Hamilton,
Ontario). FIG. 22A shows a cross-section of the trench while FIG.
22B shows a different cross-section of the trench filled with NbFeB
powder in a polyimide resin. The NbFeB powder was about 6 .mu.m in
diameter.
In another embodiment, the silicon wafer is polished on both sides
and a trench is etched on one side and filled with magnetic
particle material in a binder matrix. Sensor manufacturing
processes in accordance with U.S. Pat. Nos. 7,419,821 and 7,723,099
(referenced above) can then be performed on the other side of the
wafer. This approach has the advantage of starting the electrode
part of the sensor processes on a pristine flat surface. The binder
matrix deposition step of the magnetic zone can optionally be
performed as the last step in the overall process.
D. Hybrid Immunoassay
In a hybrid test embodiment of the present invention, a current
commercial cTnI assay (e.g., i-STAT cTnI cartridge) can be modified
to include a separate but analytically integrated magnetic capture
immunosensor. The prior art sensor covers the detection range of
0.20 ng/mL to 36.00 ng/mL, while certain embodiments of the
magnetic capture immunosensor of the present invention cover a
lower, but overlapping range of 0.002 ng/mL to 1.0 ng/mL. One such
embodiment is shown in FIG. 23, where the magnetic zone is
comprised a screen printed line of NdFeB powder in a polyimide
matrix. Another embodiment is shown in FIG. 24, where the magnetic
zone is comprised of a bulk NdFeB magnet having dimensions of 1.5
mm.times.100 .mu.m.times.40 .mu.m. Although the magnet appears to
be positioned on the front side of the chip, it is truly positioned
on the backside, and FIG. 24 is meant as a composite to show how
the magnet and electrode are aligned. FIG. 25 depicts yet other
exemplary combined sensor design where the open circles on the chip
are potential print locations for the reagents of the present
invention.
In one embodiment of the multiple amperometric magnetic
immunosensor format, the background subtraction disclosed in
jointly-owned U.S. Pat. No. 7,723,099 to Miller et al. is
performed. U.S. Pat. No. 7,723,099 is hereby incorporated by
reference in its entirety. This approach is optionally applied to
both sensors, based on the inclusion of a reference immunosensor
for the magnetic bar design. In an exemplary embodiment, the
crossover from primary use of the magnetic sensor to the standard
sensor enables a broad analytical range (e.g., from about 50 ng/mL
to about 0.001 ng/mL for cTnI).
In a preferred embodiment, the use of the overlapping range sensors
will include an instrument error detection software protocol, which
enables an inconsistency between the cTnI sensor signals to be
detected. For example, in the presence of sufficient cTnI in a test
sample, both sensors should yield elevated signals, while in the
absence of cTnI, both sensors should yield relatively low
amperometric signals. Failure of these conditions being detected by
the operating software would indicate that the analytical result is
unreliable. Consequently, the instrument would suppresses reporting
a result and instead indicate to the user that the test should be
re-run with a new cartridge.
In yet another embodiment, the stabilization of the reagents and
mode of printing enables a quick curing matrix. Print cocktails for
the enzyme conjugate include, but are not limited to the
enzyme-labeled antibody to the analyte in a protein stabilizing
matrix of less than 30% solids and more preferably 10% or lower
solids. The printed magnetic materials of the present invention
cure rapidly (i.e., in less than about 7 days). This relatively
quick curing time has the advantage of simplifying the
manufacturing process, where a short delay between chip manufacture
and assembly into a cartridge is desirable.
IV. Oscillating Magnetic Immunoassays
While the disclosure above generally is based on the concept of a
static magnetic field localized in the region of the immunosensor,
an alternative methodology is also envisaged where an oscillating
magnetic field is used adjacent to the immunosensor.
One oscillating magnetic field embodiment makes critical use of
labeled magnetically susceptible beads containing both a label
antibody, and an enzyme or fluorescent marker or any other
detectable label, termed herein as a "signaling moiety." The label
may be detected by any means known in the art including simple
microscopy or a reflectance measurement for the beads attached to
the capture site using an immunosensing device. In addition, the
label can be measured optically through a simple optical density
measurement on the capture regions or electrochemically, e.g.,
through an enzymatic reaction. Additional detection techniques
include optical resonators, nuclear magnetic resonance (NMR),
piezoelectric, pyroelectric, fluorescence, chemiluminescence and
surface acoustic wave, among others.
Another embodiment of the invention is to a method of performing a
sandwich immunoassay for an analyte in a sample with an
immunosensor on a substantially planar surface using a means for
applying an oscillating magnetic field (e.g., an electromagnet
and/or a moving fixed magnet with respect to the surface of the
immunosensor). The first step comprises mixing the magnetically
susceptible beads with a sample containing or suspected of
containing an analyte, wherein the beads are coated with an
antibody to the analyte and a signal generating moiety. The second
step requires oscillating the beads across the surface of the
immunosensor coated with a second antibody to the analyte, using
one of the magnetic means mentioned above. An antibody sandwich is
formed thereby immobilizing the beads on the immunosensor. The
sample is then washed from the immunosensor, and the signaling
moiety on the immunosensor is detected.
Various embodiments of the invention are directed to use of the
movement of the magnetically susceptible beads along a surface to
accelerate the signal generation of an immunoassay. In one
embodiment, the beads are initially located in a region between two
spaced magnetic zones and can move freely between the two.
Oscillation of the magnetic field may start slowly and increase in
frequency and the magnetically susceptible beads are forced to move
back and forth across a surface as the field changes. Through this
motion, the beads can be captured in the intervening space on a
capture area of the immunosensor having capture antibodies for a
particular target analyte. In a preferred embodiment, the beads are
labeled with analyte specific antibody or antibodies.
FIG. 26 is a schematic of an oscillating bead immunoassay (OBIA)
with a central immunosensor flanked by two adjacent magnetic zones
with the magnetically susceptible beads moving there between, and
FIG. 27 shows the enhanced degree of bead capture with time in
accordance with one embodiment of the present invention. These
immunoassay embodiments are amenable to multiplexing, i.e., testing
for more than one type of analyte, because the capture event is
localized by the capture immunosensor's specificity to a particular
analyte. FIG. 28 illustrates a multiplexed OBIA where several
different types of analyte-capturing beads are present, but where
they are effectively separated onto their individual capture sites
(e.g., separate immunosensors).
As shown in FIG. 28, the format of the OBIA can be multiplexed with
the specific capture regions for the different analytes to be
measured being arranged sequentially between the two magnetic
concentration areas. A reference sensor (not shown) is also readily
applied in this format by having a non-specific antibody layer with
the series of specific analyte capture sites. In this embodiment,
the magnetically susceptible beads are prepared containing label
antibodies thereon and are mixed together in controlled ratios,
depending on the analytical considerations of the particular assays
within the multiplexed immunoassay cartridge. All of the labeled
magnetically susceptible beads will then move between the two
magnet contact zones across each of the specific capture regions.
The different analyte-specific magnetically susceptible beads may
have the same label type or, more preferably, will have different
types of labels. Optionally, the different analyte-specific
magnetically susceptible beads can be mixed into the same sample
and allowed to react prior to their attraction to the sensor
surface via the positioning of the magnets.
Those skilled in the immunosensing art will recognize that this
assay format relies on the capture of the beads as they traverse
over the surface in the presence of the magnet fields generated on
either side of the capture areas. As such, the sensor
signal-to-noise ratio is dependent on making non-specific binding
minimal on the surface except where the specific capture antibodies
are deposited. An important feature of one embodiment of the
invention is, therefore, to employ pairs of immunosensors (e.g.,
electrodes) with indifferent antibodies where the second one acts
as a reference sensor. Here, the second sensor signal is subtracted
from the first one of the pair. This general concept is disclosed
in jointly-owned U.S. Pat. No. 7,723,099 (referenced above).
In another variant of the present embodiment, the oscillation of
the beads is kept to one side of the capture region for a period of
time in order to allow analyte capture on the immunosensor surface.
A further refinement includes a reference sensor to determine the
portion of the non-specific signal generated during the analyte
capture part of the assay. The magnetic field is preferably
actuated via a coil around two ends of a tube or conduit, similar
to, for example, the formation of NMR shim fields. This allows
control of the force the magnetically susceptible beads experience
in contacting the surface of the sensor and minimizes nonspecific
binding. Here, the coils have several axes to allow x/y and z
motion in the conduit. Fixed magnets on a rotating or oscillating
platform may be used in alternative embodiments.
EXAMPLES
The present invention will be better understood with reference to
the specific embodiments set forth in the following non-limiting
examples.
Example 1
Immunoassay for Determination of Troponin I (TnI)
FIG. 29 illustrates a comparative amperometric immunoassay for the
determination of troponin I (TnI) 70, a marker of cardiac injury.
In one embodiment, a blood sample, for example, is introduced into
the sample holding chamber of the immunosensing device of the
present invention, and is amended by a conjugate molecule 71
comprising alkaline phosphatase enzyme (AP) covalently attached to
a polyclonal anti-troponin I antibody (cTnI). This conjugate
specifically binds to the TnI 70 in the blood sample, producing a
complex made up of TnI bound to the AP-aTnI conjugate 72. The blood
sample is further amended with polymer beads with a ferrite core 74
coated with a TnI antibody. The mixture is oscillated in a conduit
connected to the holding chamber that generates sandwich formation
on the bead.
Positioned in the conduit is the sensor chip (or chips), which
includes a conductivity sensor used to monitor where the sample is
with respect to the sensor chip. The position of the sample segment
within the conduit can be actively controlled using the edge of the
fluid as a marker. As the sample/air interface crosses the
conductivity sensor, a precise signal is generated that can be used
as a fluid position marker from which controlled fluid excursions
can be executed. The fluid segment is preferentially oscillated
edge-to-edge over the sensor. The immunosensor chip is positioned
downstream of the mean oscillation position in the conduit. A bulk
magnet 76 is positioned under the sensor chip and draws the
magnetically susceptible beads to the immunosensor surface.
In the present example, the sensor comprises an amperometric
electrode used to detect the enzymatically produced 4-aminophenol
from the reaction of 4-aminophenylphosphate with the enzyme label
alkaline phosphatase. The electrode is preferably produced from a
gold surface coated with a photodefined layer of polyimide.
Regularly spaced openings in the insulating polyimide layer define
a grid of small gold electrodes at which the 4-aminophenol is
oxidized in a 2 electron per molecule reaction.
Substrates, such as p-aminophenol species, can be selected such
that the half-wave potential (E.sub.1/2) of the substrate and
product differ substantially. Preferably, the E.sub.1/2 of the
substrate is substantially higher (i.e., more positive) than that
of the product. When this condition is met, the product can be
selectively electrochemically measured in the presence of the
substrate.
The detection of alkaline phosphatase activity in this example
relies on a measurement of the 4-aminophenol oxidation current.
This is achieved at a potential of about +60 mV versus the Ag/AgCl
reference electrode on the chip. The specific form of detection
used depends on the sensor configuration. The concentration of the
4-aminophenylphosphate is selected to be in excess, e.g., 10 times
the Km value. The analysis solution is 0.1 M in diethanolamine and
1.0 M NaCl, buffered to a pH of 9.8. Additionally, the analysis
solution contains 0.5 mM MgCl, which is a cofactor for the enzyme.
A carbonate buffer may alternatively be utilized.
In various embodiments, the antibodies are selected to bind one or
more of protein, e.g., human chorionic gonadotrophin, troponin I,
troponin T, troponin C, a troponin complex, creatine kinase,
creatine kinase subunit M, creatine kinase subunit B, myoglobin,
myosin light chain, or modified fragments thereof. Such modified
fragments are generated by oxidation, reduction, deletion, addition
or modification of at least one amino acid, including chemical
modification with a natural moiety or with a synthetic moiety.
Preferably, these biomolecules bind to the analyte specifically and
have an affinity constant for binding of about 10.sup.7 to
10.sup.15 M.sup.-1.
The immunosensor was prepared as follows. A silicon wafer was
thermally oxidized to form an insulating oxide layer with a
thickness of about 1 .mu.m. A titanium/tungsten layer was sputtered
onto the oxide layer to a preferable thickness of about 100 .ANG.
to about 1000 .ANG., followed by a layer of gold that is most
preferably about 800 .ANG. thick. A photoresist was then spin
coated onto the wafer and was dried and baked. The surface was then
exposed using a contact mask, and the latent image was developed.
Next, the wafer was exposed to a gold-etchant. The patterned gold
layer was coated with a photodefinable polyimide, suitably baked,
exposed using a contact mask, developed, cleaned in an O.sub.2
plasma, and preferably imidized at 350.degree. C. for 5 hours. An
optional metallization of the back side of the wafer may be
performed to act as a resistive heating element, such as for
example in embodiments where the immunosensor is to be used in a
thermostatted format.
Example 2
Magnetic Immunosensing Device and Method of Use
The present example describes a method of using a magnetic
immunosensing device in accordance with one embodiment of the
invention. As shown in FIGS. 30-33, an unmetered fluid sample was
introduced into sample chamber 34 of a cartridge, through a sample
entry port 4. Capillary stop 25 prevents passage of the sample into
conduit 11 at this stage, and conduit 34 is filled with the sample.
Lid 2 is closed to prevent leakage of the sample from out of the
cartridge. The cartridge is then inserted into a reading apparatus,
such as that disclosed in U.S. Pat. No. 5,821,399 to Zelin
(referenced above), which is hereby incorporated by reference.
Insertion of the cartridge into a reading apparatus activates the
mechanism which punctures a fluid-containing package located at 42
when the package is pressed against spike 38. Fluid is thereby
expelled into the second conduit, arriving in sequence at 39, 20,
12 and 11. The constriction at 12 prevents further movement of
fluid because residual hydrostatic pressure is dissipated by the
flow of fluid via second conduit portion 11 into the waste chamber
44. In a second step, operation of a pump means applies pressure to
air-bladder 43, forcing air through conduit 40, through cutaways 17
and 18, and into conduit 34 at a predetermined location 27.
Capillary stop 25 and location 27 delimit a metered portion of the
original sample. While the sample is within sample chamber 34, it
is optionally amended with a compound or compounds present
initially as a dry coating on the inner surface of the chamber
(e.g., antibody-coated magnetically susceptible beads and
enzyme-labeled antibody conjugate). The metered portion of the
sample is then expelled through the capillary stop by air pressure
produced within air-bladder 43. The sample optionally is oscillated
in order to promote efficient sandwich formation on the
magnetically susceptible beads. Preferably, an oscillation
frequency of between about 0.2 Hz and about 5 Hz is used, most
preferably about 0.7 Hz.
In the next step, the sample is moved forwards along the conduit
such that the magnetically susceptible beads can become trapped
onto the surface of the magnetic electrode. Subsequently, the
sample is ejected from the conduit by further pressure applied to
air-bladder 43, and the sample passes to waste chamber 44. A wash
step next removes non-specifically bound enzyme-conjugate from the
immunosensor area of the conduit. Wash fluid in the second conduit
is moved by a pump means 43, into contact with the sensors.
The air segment (meniscus) or segments are produced within a
conduit by any suitable means, including a passive means, an
embodiment of which is shown in FIG. 34 and described in detail in
U.S. Pat. No. 7,682,833 (referenced above), or an active means
including a transient lowering of the pressure within a conduit
using pump means whereby air is drawn into the conduit through a
flap or valve. The air segment is extremely effective at clearing
the sample-contaminated fluid from conduit 15. The efficiency of
the rinsing of the sensor region is greatly enhanced by the
introduction of one or more air segments. The leading and/or
trailing edges of air segments are passed one or more times over
the sensors to rinse and resuspend extraneous material that may
have been deposited from the sample. Extraneous material includes
any material other than specifically bound analyte or
analyte/antibody-enzyme conjugate complex. However, in accordance
with various embodiments of the invention, the washing or rinsing
step is not sufficiently protracted or vigorous so as to promote
substantial resuspension of the magnetically susceptible beads or
dissociation of specifically bound analyte or
analyte/antibody-enzyme conjugate complex from the beads. For
measurement, a further portion of fluid containing the enzyme
substrate is placed over the beads on the immunosensors, and the
current or potential, as appropriate to the mode of operation, is
recorded as a function of time.
FIG. 34 illustrates the construction of a specific means for
passively introducing an air segment into the sample fluid. Within
the base of the immunosensor is recess 140 comprising a tapered
portion 141 and a cylindrical portion 142 that are connected. The
tapered portion is in fluid connection with a hole of similar
diameter in the tape gasket (FIG. 32) that separates the base (FIG.
33) and cover (FIGS. 30 and 31) of the assembled immunosensor
cartridge. The recess contains an absorbent material that, upon
contact with fluid, withdraws a small quantity of fluid from a
conduit thereby passively introducing an air segment into the
conduit. The volume of the recess and the amount and type of
material within it may be adjusted to control the size of the air
segment introduced. Specific absorbent materials include, but are
not limited to, glass filter and a laminate comprising a 3 .mu.m
Versapor.RTM. filter (i.e., acrylic copolymer membrane cast on a
nonwoven nylon support) bonded by sucrose to a 60% viscose chiffon
layer.
Example 3
Magnetic Immunosensing Device and Method of Use
The present example describes one of the methods of use of a
cartridge. In this embodiment, the cartridge includes a closeable
valve, located between the immunosensor and the waste chamber. For
a cTnI assay, a blood sample is first introduced into the sample
chamber of the cartridge. In the following time sequence, time zero
(t=0) represents the time at which the cartridge is inserted into
the cartridge reading device. Times are given in minutes. Between
t=0 and t=1.5, the cartridge reading device makes electrical
contact with the sensors through electrical contact pads and
performs certain diagnostic tests. Insertion of the cartridge
perforates the foil pouch introducing fluid into the second
conduit, as previously described herein. The diagnostic tests
determine whether fluid or sample is present in the conduits using
the conductivity electrodes, determine whether electrical short
circuits are present in the electrodes, and ensure that the sensor
and ground (e.g., reference/counter) electrodes are thermally
equilibrated to, preferably, 37.degree. C. prior to the analyte
determination.
Between t=1.5 and t=6.75, a metered portion of the sample,
preferably between about 4 .mu.L and about 200 .mu.L, more
preferably between about 4 .mu.L and about 20 .mu.L, and most
preferably about 7 .mu.L, is used to contact the sensor. The edges
defining the forward and trailing edges of the sample are
reciprocally moved over the conductivity sensor region at a
frequency that is preferably between 0.2 to 5.0 Hz, and is most
preferably 0.7 Hz. During this time, the enzyme-antibody conjugate
and magnetically susceptible beads dissolve within the sample. The
amount of enzyme-antibody conjugate that is coated onto the conduit
is selected to yield a concentration when dissolved that is
preferably higher than the highest anticipated cTnI concentration,
and is most preferably six times higher than the highest
anticipated cTnI concentration in the sample.
Between t=6.75 and t=10.0, the sample is moved to the immunosensor
for capture of the magnetically susceptible beads. As shown in
FIGS. 30-33, the sample is moved into the waste chamber via
closeable valve 41, wetting the closeable valve and causing it to
close. The seal created by the closing of the valve 41 permits the
first pump means to be used to control motion of fluid from conduit
11 to conduit 15. After the valve 41 closes and the remaining
sample is locked in the post analysis conduit, the analyzer plunger
retracts from the flexible diaphragm of the pump mean, creating a
partial vacuum in the sensor conduit. This forces the analysis
fluid through the small hole in the tape gasket 31 and into a short
transecting conduit in the base, 13 and 14. The analysis fluid is
then pulled further and the front edge of the analysis fluid is
oscillated across the surface of the immunosensor chip in order to
shear the sample near the walls of the conduit. The conductivity
sensor on the chip is used to control this process.
The efficiency of the wash is optimally further enhanced by
introduction into the fluid of one or more meniscus or air segment.
As previously described, the air segment may be introduced by
either active or passive means. Fluid is then forcibly moved
towards sensor chip by the partial vacuum generated by reducing the
mechanical pressure exerted upon paddle 6, causing the "T" region
of the sensor channel in the vicinity of the transecting conduit to
fill with analysis fluid. The T region of the sensor channel
optionally has a higher channel height resulting in a meniscus with
a smaller radius of curvature. Further away from the T region
towards the post-analytical conduit, the conduit height is
optionally smaller. The analysis fluid passively flows from the T
region towards this low conduit height region, thereby washing the
conduit walls. This passive leak allows further effective washing
of the T region using a minimal volume of fluid and without
displacing the magnetically susceptible beads. In this embodiment,
the fluid located within the second conduit also contains a
substrate for the enzyme. In other embodiments, amendment of the
fluid using dried substrate within the second conduit may be
utilized.
Following the positioning of a final segment of fluid over the
sensor, measurement of the sensor response is recorded and the
concentration of analyte is determined. Specifically, at least one
sensor reading of a sample is made by rapidly placing over the
sensor a fresh portion of fluid containing a substrate for the
enzyme. Rapid displacement both rinses away product previously
formed, and provides a new substrate to the electrode. Repetitive
signals are averaged to produce a measurement of higher precision,
and also to obtain a better statistical average of the baseline,
represented by the current immediately following replacement of the
solution over the immunosensor.
Example 4
Magnetic Immunosensing Device
Referring now to FIG. 35, there is shown a top view of a magnetic
immunosensor cartridge in accordance with one embodiment of the
present invention. Cartridge 150 comprises a base and a top
portion, preferably constructed of plastic. The two portions are
connected by a thin, adhesive gasket or thin pliable film. As in
previous embodiments, the assembled cartridge comprises a sample
chamber 151 into which a sample containing an analyte of interest
is introduced via sample inlet 152. A metered portion of the sample
is delivered to the sensor chip 153 (comprising an integrated
magnetic layer) via the sample conduit 154 (first conduit) by the
combined action of a capillary stop 152, preferably formed by a
0.012'' laser cut hole in the gasket or film that connects the two
portions of the cartridge, and an entry point 155 located at a
predetermined point within the sample chamber whereby air is
introduced by the action of a pump means, such as a paddle pushing
upon a sample diaphragm 156. After magnetic capture of the beads on
the immunosensor, the sample is moved to vent 157, which contains a
wicking material that absorbs the sample and thereby seals the vent
closed to the further passage of liquid or air. The wicking
material is preferably a cotton fiber material, a cellulose
material, or other hydrophilic material having pores. It is
important in the present application that the material is
sufficiently absorbent (i.e., possesses sufficient wicking speed)
that the valve closes within a time period that is commensurate
with the subsequent withdrawal of the sample diaphragm actuating
means, so that the sample is not subsequently drawn back into the
region of the immunosensor.
In specific embodiments of the invention, there is provided a wash
conduit (second conduit) 158, connected at one end to a vent 159
and at the other end to the sample conduit at a point 160 of the
sample conduit that is located between vent 157 and immunosensor
chip 153. Upon insertion of the cartridge into a reading apparatus,
a fluid is introduced into conduit 158. Preferably, the fluid is
present initially within a foil pouch 161 that is punctured by a
pin when an actuating means applies pressure upon the pouch. There
is also provided a short conduit 162 that connects the fluid to
conduit 154 via a small opening in the gasket 163. A second
capillary stop initially prevents the fluid from reaching capillary
stop 160, so that the fluid is retained within conduit 158.
After vent 157 has closed, the pump means is actuated, creating a
lowered pressure within conduit 154. Air vent 164, preferably
comprising a small flap cut in the gasket or a membrane that
vibrates to provide an intermittent air stream, provides a means
for air to enter conduit 158 via a second vent 165. The second vent
165 preferably also contains wicking material capable of closing
the vent if wetted, which permits subsequent depression of sample
diaphragm 156 to close vent 165, if required. Simultaneously with
the actuation of sample diaphragm 156, fluid is drawn from conduit
158, through capillary stop 160, into conduit 154. Because the flow
of fluid is interrupted by air entering vent 164, at least one air
segment (e.g., a segment or stream of segments) is introduced.
Further withdrawal of sample diaphragm 156 draws the liquid
containing at least one air segment back across the sensing surface
of sensor chip 153. The presence of air-liquid boundaries within
the liquid enhances the rinsing of the sensor chip surface to
remove remaining sample. Preferably, the movement of the sample
diaphragm 156 is controlled in conjunction with signals received
from the conductivity electrodes housed within the sensor chip
adjacent to the analyte sensors. In this way, the presence of
liquid over the sensor is detected, and multiple readings can be
performed by movement of the fluid in discrete steps.
It is advantageous in this embodiment to perform analyte
measurements when only a thin film of fluid coats the magnetically
susceptible beads on the immunosensors, ground chip 165, and a
contiguous portion of the wall of conduit 154 between the sensors
and ground electrode. A suitable film is obtained by withdrawing
fluid by operation of the sample diaphragm 156, until the
conductimetric sensor located next to the sensor indicates that
bulk fluid is no longer present in that region of conduit 154. It
has been found that measurement can be performed at very low (nA)
currents, and the potential drop that results from increased
resistance of a thin film between ground chip and sensor chip
(compared to bulk fluid) is not significant.
The ground chip 165 is preferably a silver/silver chloride
reference electrode and acts effectively as both a counter and
reference electrode in an amperometric measurement. It is
advantageous, in this embodiment of the invention, to avoid air
segments, which easily form upon the relatively hydrophobic silver
chloride surface, to pattern the ground chip as small regions of
silver/silver chloride interspersed with more hydrophilic regions,
such as a surface of silicon dioxide. Thus, a preferred ground
electrode (counter and reference electrode combined) configuration
comprises an array of silver/silver chloride squares densely
arranged and interspersed with silicon dioxide. There is a further
advantage in the avoidance of unintentional segments if the regions
of silver/silver chloride are somewhat recessed.
Referring now to FIGS. 7 and 36, there is shown a schematic view of
the fluidics of the preferred embodiment of an immunosensor
cartridge. Regions R1-R7 represent specific regions of the conduits
associated with specific operational functions. In particular, R1
represents the sample chamber; R2 the sample conduit whereby a
metered portion of the sample is transferred to the capture region,
and in which the sample is optionally amended with a substance
coated upon the walls of the conduit (e.g., magnetically
susceptible beads with antibody to the analyte and antibody
conjugate); R21 represents a region for mixing and sample
oscillation; R3 represents the magnetic capture region, which
houses the immunosensors; R4 and R5 represent portions of the first
conduit that are optionally used for further amendment of fluids
with substances coated onto the conduit wall, whereby more complex
assay schemes are achieved; R6 represents the portion of the second
conduit into which fluid is introduced upon insertion of the
cartridge into a reading apparatus; R7 comprises a portion of the
conduit located between capillary stops 160 and 166 (shown in FIG.
35), in which further amendment can occur; and R8 represents the
portion of conduit 154 located between point 160 and vent 157,
which can further be used to amend liquids contained within.
Example 5
Magnetic Immunosensor System
This example addresses the coordination of fluidics and analyte
measurements as a system. In the analysis sequence, a user places a
sample into the cartridge, places the cartridge into the analyzer
and in about 1 minute to about 20 minutes, a quantitative
measurement of one or more analytes is performed. Referring to
FIGS. 35 and 36, the following is a non-limiting example of a
sequence of events that occur during the analysis:
1) A 25 to 50 .mu.L sample is introduced in the sample inlet 167
and fills chamber 151 to capillary stop 152 formed by a 0.012''
laser cut hole in the adhesive tape holding the cover and base
components together. The user then seals the inlet and places the
cartridge into the analyzer. Magnetically susceptible beads having
antibodies to the analyte of interest and optionally a labeled
conjugate antibody to the analyte may be provided in a dry coating
on the walls of chamber 151 such that they dissolve into the sample
once the sample is introduced therein. In an alternative
embodiment, either or both the magnetically susceptible beads and
the labeled conjugate antibody may be provided in a dry coating
within conduit 154.
2) The analyzer makes contact with the cartridge, and a motor
driven plunger presses onto the foil pouch 161, forcing the
wash/analysis fluid out into a central conduit 158.
3) A separate motor driven plunger contacts the sample diaphragm
156, pushing a measured segment of the sample along the sample
conduit (from reagent region R1 to R2 and R21). The sample position
is detected via one or more conductivity sensors. The immunosensor
chip is located in capture region R3.
4) The sample is oscillated by means of the sample diaphragm 156 in
the R21 region in a predetermined and controlled fashion for a
controlled time to promote binding of analyte to the magnetically
susceptible beads and to the antibody conjugate.
5) The sample is pushed towards the waste region of the cartridge
(R8) and comes in contact with a passive pump 157 in the form of a
cellulose or similar absorbent wick. The action of wetting this
wick seals the wick to air flow, thus eliminating its ability to
vent excess pressure generated by the sample diaphragm 156. The
active vent thereby becomes the "controlled air vent" of FIG.
36.
6) Rapid evacuation of the sample conduit (effected by withdrawing
the motor driven plunger from the sample diaphragm 156) forces a
mixture of air (from the vent) and wash/analysis fluid from the
second conduit to move into the inlet located between R5 and R4. By
repeating the rapid evacuation of the sample conduit, a series of
air separated fluid segments are generated, which are pulled across
the sensor chip towards the sample inlet (from R4 to R3 to R21 to
R2 and R1). This process washes the sensor free of excess reagents
and wets the sensor with reagents appropriate for the analysis. In
certain embodiments, the wash/analysis fluid that originates in the
foil pouch can be further amended by addition of reagents in R7 and
R6 within the central wash/analysis fluid conduit.
7) The wash/analysis fluid segment is drawn at a slower speed
towards the sample inlet to yield an immunosensor chip with the
retained magnetically susceptible beads, which contains only a thin
layer of the analysis fluid. The electrochemical analysis is
performed at this point. The preferred method of analysis is
amperometry, but potentiometry or impedance detection is also
used.
8) The mechanism retracts, allowing the cartridge to be removed
from the analyzer.
While the present invention has been described in terms of various
preferred embodiments, those skilled in the art will recognize that
modifications, substitutions, omissions and changes can be made to
such embodiments without departing from the spirit and scope of the
present invention.
* * * * *